Humanization of rabbit antibodies using a universal antibody framework

ABSTRACT

The present invention relates to an universal antibody acceptor framework and to methods for grafting non-human antibodies, e.g., rabbit antibodies, using a universal antibody acceptor framework. Antibodies generated by the methods of the invention are useful in a variety of diagnostic and therapeutic applications.

RELATED INFORMATION

The present application is a divisional application of U.S. applicationSer. No. 14/179,141 filed Feb. 12, 2014 (now allowed), which is adivisional application of U.S. application Ser. No. 13/616,214 filedSep. 14, 2012 (now U.S. Pat. No. 8,937,162), which is a divisionalapplication of U.S. application Ser. No. 13/000,309 filed Dec. 20, 2010(now U.S. Pat. No. 8,293,235), which is a 371 national stage applicationof International Application No. PCT/CH2009/000222 filed Jun. 25, 2009,which claims priority to U.S. Application Ser. No. 61/075,697 filed onJun. 25, 2008, and further claims priority to U.S. Application Ser. Nos.61/155,041 and 61/155,105 filed on Feb. 24, 2009.

BACKGROUND OF THE INVENTION

Monoclonal antibodies, their conjugates and derivatives are hugelycommercially important as therapeutic and diagnostic agents. Non-humanantibodies elicit a strong immune response in patients, usuallyfollowing a single low dose injection (Schroff, 1985 Cancer Res45:879-85, Shawler. J Immunol 1985 135:1530-5; Dillman, Cancer Biother1994 9:17-28). Accordingly, several methods for reducing theimmunogenicity of murine and other rodent antibodies as well astechnologies to make fully human antibodies using e.g. transgenic miceor phage display were developed. Chimeric antibodies were engineered,which combine rodent variable regions with human constant regions (e.g.,Boulianne Nature 1984 312:643-6) reduced immunogenicity problemsconsiderably (e.g., LoBuglio, Proc Natl Acad Sci 1989 86:4220-4; Clark,Immunol Today 2000 21:397-402). Humanized antibodies were alsoengineered, in which the rodent sequence of the variable region itselfis engineered to be as close to a human sequence as possible whilepreserving at least the original CDRs, or where the CDRs form the rodentantibody were grafted into framework of a human antibody (e.g.,Riechmann, Nature 1988 332:323-7; U.S. Pat. No. 5,693,761). Rabbitpolyclonal antibodies are widely used for biological assays such asELISAs or Western blots. Polyclonal rabbit antibodies are oftentimesfavored over polyclonal rodent antibodies because of their usually muchhigher affinity. Furthermore, rabbit oftentimes are able to elicit goodantibody responses to antigens that are poorly immunogenic in miceand/or which give not rise to good binders when used in phage display.Due to these well-known advantages of rabbit antibodies, they would beideal to be used in the discovery and development of therapeuticantibodies. The reason that this is not commonly done is mainly due totechnical challenges in the generation of monoclonal rabbit antibodies.Since myeloma-like tumors are unknown in rabbits, the conventionalhybridoma technology to generate monoclonal antibodies is not applicableto rabbit antibodies. Pioneering work in providing fusion cell linepartners for rabbit antibody-expressing cells has been done by Knightand colleagues (Spieker-Polet et al., PNAS 1995, 92:9348-52) and animproved fusion partner cell line has been described by Pytela et al. in2005 (see e.g. U.S. Pat. No. 7,429,487). This technology, however, isnot wildly spread since the corresponding know-how is basicallycontrolled by a single research group. Alternative methods for thegeneration of monoclonal antibodies that involve the cloning ofantibodies from selected antibody-expressing cells via RT-PCR aredescribed in the literature, but have never been successfully reportedfor rabbit antibodies.

Rabbit antibodies, like mouse antibodies are expected to elicit strongimmune responses if used for human therapy, thus, rabbit antibodies needto be humanized before they can be used clinically. However, the methodsthat are used to make humanized rodent antibodies cannot easily beextrapolated for rabbit antibodies due to structural differences betweenrabbit and mouse and, respectively, between rabbit and human antibodies.For example, the light chain CDR3 (CDRL3) is often much longer thanpreviously known CDRL3s from human or mouse antibodies.

There are few rabbit antibody humanization approaches described in theprior art which are, however, no classical grafting approach in whichthe CDRs of a non-human donor are transplanted on a human acceptorantibody. WO 04/016740 describes a so-called “resurfacing” strategy. Thegoal of a “resurfacing” strategy is to remodel the solvent-accessibleresidues of the non-human framework such that they become morehuman-like. Similar humanization techniques for rabbit antibodies asdescribed in WO 04/016740 are known in the art. Both WO08/144757 andWO05/016950 discloses methods for humanizing a rabbit monoclonalantibody which involve the comparison of amino acid sequences of aparent rabbit antibody to the amino acid sequences of a similar humanantibody. Subsequently, the amino acid sequence of the parent rabbitantibody is altered such that its framework regions are more similar insequence to the equivalent framework regions of the similar humanantibody. In order to gain good binding capacities, laboriousdevelopment efforts need to be made for each immunobinder individually.

A potential problem of the above-described approaches is that not ahuman framework is used, but the rabbit framework is engineered suchthat it looks more human-like. Such approach carries the risk that aminoacid stretches that are buried in the core of the protein still mightcomprise immunogenic T cell epitopes.

To date, the applicants have not identified a rabbit antibody, which washumanized by applying state-of-the-art grafting approaches. This mightbe explained by fact that rabbit CDRs may be quite different from humanor rodent CDRs. As known in the art, many rabbit VH chains have extrapaired cysteines relative to the murine and human counterparts. Inaddition to the conserved disulfide bridge formed between cys22 andcys92, there is also a cys21-cys79 bridge as well as an interCDR S—Sbridge formed between the last residue of CDRH1 and the first residue ofCDR H2 in some rabbit chains. Besides, pairs of cysteine residues areoften found in the CDR-L3. Moreover, many rabbit antibody CDRs do notbelong to any previously known canonical structure. In particular theCDR-L3 is often much longer than the CDR-L3 of a human or murinecounterpart.

Hence, the grafting of non-human CDRs antibodies into a human frameworkis a major protein engineering task. The transfer of antigen bindingloops from a naturally evolved framework to a different artificiallyselected human framework must be performed so that native loopconformations are retained for antigen binding. Often antigen bindingaffinity is greatly reduced or abolished after loop grafting. The use ofcarefully selected human frameworks in grafting the antigen bindingloops maximizes the probability of retaining binding affinity in thehumanized molecule (Roguzka et al 1996). Although the many graftingexperiments available in the literature provide a rough guide for CDRgrafting, it is not possible to generalize a pattern. Typical problemsconsist in losing the specificity, stability or producibility aftergrafting the CDR loops.

Accordingly, there is an urgent need for improved methods for reliablyand rapidly humanizing rabbit antibodies for use as therapeutic anddiagnostic agents. Furthermore, there is a need for human acceptorframeworks for reliably humanizing rabbit antibodies, providingfunctional antibodies and/or antibody fragments with drug-likebiophysical properties.

SUMMARY OF THE INVENTION

It has surprisingly been found that a highly soluble and stable humanantibody framework identified by a Quality Control (QC) assay (asdisclosed in WO 0148017 and in Auf der Maur et al (2001), FEBS Lett 508,p. 407-412) is particularly suitable for accommodating CDRs from othernon-human animal species, for example, rabbit CDRs. Accordingly, in afirst aspect, the invention provides the light and heavy chain variableregions of a particular human antibody (the so called, “FW 1.4”antibody) which is especially suitable as universal acceptor for CDRsfrom a variety of antibodies, in particular from rabbit antibodies, ofdifferent binding specificities, independent of whether a disulfidebridge is present in a CDR or not. Furthermore, the present inventionprovides two mutant sequences of said particular human antibodyframework, namely rFW1.4 and rFW1.4(V2), both being frameworksparticularly suitable as universal acceptor frameworks for the graftingof rabbit CDRs. In another aspect, the invention provides a motif offramework residues which renders a human framework suitable foraccommodating CDRs from other non-human animal species, in particularrabbit CDRs.

Humanized immunobinders generated by the grafting of rabbit CDRs intothese highly compatible variable light and heavy chain frameworksconsistently and reliably retain the spatial orientation of the rabbitantibodies from which the donor CDRs are derived. Therefore, nostructurally relevant positions of the donor immunobinder need to beintroduced into the acceptor framework. Due to these advantages,high-throughput humanization of rabbit antibodies with no or littleoptimization of the binding capacities can be achieved.

Accordingly, in another aspect, the invention provides methods forgrafting rabbit and other non-human CDRs, into the soluble and stablelight chain and/or heavy chain human antibody framework sequencesdisclosed herein, thereby generating humanized antibodies with superiorbiophysical properties. In particular, immunobinders generated by themethods of the invention exhibit superior functional properties such assolubility and stability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the CDR H1 definition used herein for grafting antigenbinding sites from rabbit monoclonal antibodies into the highly solubleand stable human antibody frameworks.

FIG. 2 schematically shows a B-cell 1 labeled with a fluorescentantibody 2 interacting with a target-expressing cell 3 stained with anintracellular dye 4. Target of choice: 5; BCR: 6.

FIGS. 3A, 3B, and 3C: the FACS selection process of rabbit B cellsbinding to ESBA903 soluble target. FIG. 3A: Lymphocytes are gatedaccording to forward and side scatter. FIG. 3B: Among them, IgG+ IgM−cells (probably memory B cells) are selected (red gate). FIG. 3C: Cellsdouble-stained with ESBA903-PE and ESBA903-PerCP (green gate) areexpected to encode high affinity IgGs against ESBA903. Cells showing thebrightest fluorescence (pink gate) were sorted in 96-well plates.

FIG. 4. Beads coated with anti-TNFalpha antibodies (PE labeled) bind toTNFalpha-transfected CHO cells (upper panel). Control beads coatedanti-CD19 antibodies (APC labeled) do not bind TNFalpha transfected CHOcells (middle panel). Beads coated with anti-TNFalpha antibodies (PElabeled) do not bind to wildtype (wt) CHO cells (lower panel). Dot plotson the left show forward and side scatters, which indicate respectivelythe size and the granularity of the events. Single beads (˜3 um)population is gated in P2. CHO cells eventually bound to beads (˜30 um)are gated in P1. Dot plots in the middle show the P1 events (CHO cells)in respect to their PE or APC staining. Thus, if cells interact withanti-TNFalpha beads, they will be shown in P3 gate, and if they interactwith the anti-CD19 beads they will appear in P4 gate. On the right,statistics for each sample are detailed.

FIG. 5. Beads coated with anti-TNFalpha-PE and beads coated withanti-CD19-APC were mixed together with TNFalpha-transfected CHO cells.CHO cells were gated (P1) and among them cells binding to eitheranti-TNFalphaPE coated beads or anti-CD19-APC coated beads are shown ingates P3 and P4, respectively. Unbound beads are visible in gate P2.

FIG. 6. An analysis of rabbit antibody sequences extracted from theKabat database confirms that CDR3 of the variable heavy chain istypically by three amino acids longer than its murine counterpart.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In order that the present invention may be more readily understood,certain terms will be defined as follows. Additional definitions are setforth throughout the detailed description.

The term “antibody” refers to whole antibodies and any antigen bindingfragment. The term “antigen binding polypeptide” and “immunobinder” areused simultaneously herein. An “antibody” refers to a protein,optionally glycosylated, comprising at least two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, or an antigenbinding portion thereof. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as V_(H)) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as V_(L)) and a light chain constantregion. The light chain constant region is comprised of one domain, CL.The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”) refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., TNF). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of theV_(L), V_(H), CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the V_(H) and CH1domains; (iv) a Fv fragment consisting of the V_(L) and V_(H) domains ofa single arm of an antibody, (v) a single domain or dAb fragment (Wardet al., (1989) Nature 341:544-546), which consists of a V_(H) domain;and (vi) an isolated complementarity determining region (CDR) or (vii) acombination of two or more isolated CDRs which may optionally be joinedby a synthetic linker. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies. Antigen-binding portions can be produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact immunoglobulins. Antibodies can be of different isotype, forexample, an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1,IgA2, IgD, IgE, or IgM antibody.

The term “immunobinder” refers to a molecule that contains all or a partof the antigen binding site of an antibody, e.g. all or part of theheavy and/or light chain variable domain, such that the immunobinderspecifically recognizes a target antigen. Non-limiting examples ofimmunobinders include full-length immunoglobulin molecules and scFvs, aswell as antibody fragments, including but not limited to (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fab′ fragment, which is essentially a Fab with part ofthe hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed.1993); (iv) a Fd fragment consisting of the V_(H) and C_(H)1 domains;(v) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (vi) a single domain antibody such as a Dab fragment(Ward et al., (1989) Nature 341:544-546), which consists of a V_(H) orV_(L) domain, a Camelid (see Hamers-Casterman, et al., Nature363:446-448 (1993), and Dumoulin, et al., Protein Science 11:500-515(2002)) or a Shark antibody (e.g., shark Ig-NARs Nanobodies®; and (vii)a nanobody, a heavy chain variable region containing a single variabledomain and two constant domains.

The term “single chain antibody”, “single chain Fv” or “scFv” is refersto a molecule comprising an antibody heavy chain variable domain (orregion; V_(H)) and an antibody light chain variable domain (or region;V_(L)) connected by a linker. Such scFv molecules can have the generalstructures: NH₂-V_(L)-linker-V_(H)-COOH or NH₂-V_(H)-linker-V_(L)-COOH.A suitable state of the art linker consists of repeated GGGGS (SEQ IDNO: 26) amino acid sequences or variants thereof. In a preferredembodiment of the present invention a (GGGGS)₄ linker of the amino acidsequence set forth in SEQ ID NO: 8 is used, but variants of 1-3 repeatsare also possible (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA90:6444-6448). Other linkers that can be used for the present inventionare described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi etal. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res.56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 andRoovers et al. (2001), Cancer Immunol.

As used herein, the term “functional property” is a property of apolypeptide (e.g., an immunobinder) for which an improvement (e.g.,relative to a conventional polypeptide) is desirable and/or advantageousto one of skill in the art, e.g., in order to improve the manufacturingproperties or therapeutic efficacy of the polypeptide. In oneembodiment, the functional property is stability (e.g., thermalstability). In another embodiment, the functional property is solubility(e.g., under cellular conditions). In yet another embodiment, thefunctional property is aggregation behavior. In still anotherembodiment, the functional property is protein expression (e.g., in aprokaryotic cell). In yet another embodiment the functional property isrefolding behavior following inclusion body solubilization in amanufacturing process. In certain embodiments, the functional propertyis not an improvement in antigen binding affinity. In another preferredembodiment, the improvement of one or more functional properties has nosubstantial effect on the binding affinity of the immunobinder.

The term “CDR” refers to one of the six hypervariable regions within thevariable domains of an antibody that mainly contribute to antigenbinding. One of the most commonly used definitions for the six CDRs wasprovided by Kabat E. A. et al., (1991) Sequences of proteins ofimmunological interest. NIH Publication 91-3242). As used herein,Kabat's definition of CDRs only apply for CDR1, CDR2 and CDR3 of thelight chain variable domain (CDR L1, CDR L2, CDR L3, or L1, L2, L3), aswell as for CDR2 and CDR3 of the heavy chain variable domain (CDR H2,CDR H3, or H2, H3). CDR1 of the heavy chain variable domain (CDR H1 orH1), however, as used herein is defined by the residue positions (Kabatnumbering) starting with position 26 and ending prior to position 36.This definition is basically a fusion of CDR H1 as differently definedby Kabat and Chotia (see also FIG. 1 for illustration).

The term “antibody framework” as used herein refers to the part of thevariable domain, either VL or VH, which serves as a scaffold for theantigen binding loops (CDRs) of this variable domain. In essence it isthe variable domain without the CDRs.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds (e.g.,a specific site on the TNF molecule). An epitope typically includes atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive ornon-consecutive amino acids in a unique spatial conformation. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

The terms “specific binding,” “selective binding,” “selectively binds,”and “specifically binds,” refer to antibody binding to an epitope on apredetermined antigen. Typically, the antibody binds with an affinity(K_(D)) of approximately less than 10⁻⁷ M, such as approximately lessthan 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower.

The term “K_(ID)” or “Kd” refers to the dissociation equilibriumconstant of a particular antibody-antigen interaction. Typically, theantibodies of the invention bind to TNF with a dissociation equilibriumconstant (K_(D)) of less than approximately 10⁻⁷ M, such as less thanapproximately 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower, for example, asdetermined using surface plasmon resonance (SPR) technology in a BIACOREinstrument.

The term “nucleic acid molecule,” as used herein refers to DNA moleculesand RNA molecules. A nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA. A nucleic acidis “operably linked” when it is placed into a functional relationshipwith another nucleic acid sequence. For instance, a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence.

The term “vector,” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. In oneembodiment, the vector is a “plasmid,” which refers to a circular doublestranded DNA loop into which additional DNA segments may be ligated. Inanother embodiment, the vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome. The vectors disclosedherein can be capable of autonomous replication in a host cell intowhich they are introduced (e.g., bacterial vectors having a bacterialorigin of replication and episomal mammalian vectors) or can be can beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome (e.g.,non-episomal mammalian vectors).

The term “host cell” refers to a cell into which and expression vectorhas been introduced. Host cells include bacterial, microbial, plant oranimal cells, preferably, Escherichia coli, Bacillus subtilis;Saccharomyces cerevisiae, Pichia pastoris, CHO (Chinese Hamster Ovarylines) or NS0 cells.

The term “lagomorphs” refers to members of the taxonomic orderLagomorpha, comprising the families Leporidae (e.g. hares and rabbits),and the Ochotonidae (pikas). In a most preferred embodiment, thelagomorphs is a rabbit. The term “rabbit” as used herein refers to ananimal belonging to the family of the leporidae.

As used herein, “identity” refers to the sequence matching between twopolypeptides, molecules or between two nucleic acids. When a position inboth of the two compared sequences is occupied by the same base or aminoacid monomer subunit (for instance, if a position in each of twopolypeptides is occupied by a lysine), then the respective molecules areidentical at that position. The “percentage identity” between twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences. Generally, a comparison is made when two sequences arealigned to give maximum identity. Such alignment can be provided using,for instance, the method of the Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) algorithm which has been incorporated into the GAPprogram in the GCG software package, using either a Blossum 62 matrix ora PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Various aspects of the invention are described in further detail in thefollowing subsections. It is understood that the various embodiments,preferences and ranges may be combined at will. Further, depending ofthe specific embodiment, selected definitions, embodiments or ranges maynot apply.

If not otherwise stated, the amino acid positions are indicatedaccording to the AHo numbering scheme. The AHo numbering system isdescribed further in Honegger, A. and Pluckthun, A. (2001) J Mol. Biol.309:657-670). Alternatively, the Kabat numbering system as describedfurther in Kabat et al. (Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) may be used.Conversion tables for the two different numbering systems used toidentify amino acid residue positions in antibody heavy and light chainvariable regions are provided in A. Honegger, J. Mol. Biol. 309 (2001)657-670.

In a first aspect, the present invention provides a universal acceptorframework for the grafting of CDRs from other animal species, forexample, from rabbit. It has previously been described that antibodiesor antibody derivatives comprising the human frameworks identified inthe so called “Quality Control” screen (WO0148017) are characterised bya generally high stability and/or solubility. Although the humansingle-chain framework FW1.4 (a combination of SEQ ID NO: 1 (named a43in WO03/097697) and SEQ ID NO: 2 (named KI27 in WO03/097697)) clearlyunderperformed in the Quality Control assay, it was surprisingly foundthat it has a high intrinsic thermodynamic stability and is wellproducible, also in combination with a variety of different CDRs. Thestability of this molecule can be attributed mostly to its frameworkregions. It has further been shown that FW1.4 is in essence highlycompatible with the antigen-binding sites of rabbit antibodies.Therefore, the FW1.4 represents a suitable scaffold to construct stablehumanized scFv antibody fragments derived from grafting of rabbit loops.Thus, in one aspect, the invention provides an immunobinder acceptorframework, comprising a VH sequence having at least 90% identity to SEQID No. 1 and/or a VL sequence having at least 85% identity to SEQ ID No.2, more preferably comprising the sequence of FW1.4 (SEQ ID NO: 3) forthe grafting of rabbit CDRs, or a sequence having at least 60%, morepreferably at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, identity to SEQID NO: 3.

Moreover, it was found that FW1.4 could be optimized by substitutingseveral residue positions in the heavy chain of FW1.4 and/or bysubstituting 1 position in the light chain of FW1.4. Thereby, it wassurprisingly found that loop conformation of a large variety of rabbitCDRs in the VH could be fully maintained, largely independent of thesequence of the donor framework. Said residues in the heavy chain aswell as the 1 position in the light chain of FW1.4 are conserved inrabbit antibodies. The consensus residue for the positions in the heavychain as well as the one position in the light chain was deduced fromthe rabbit repertoire and introduced into the sequence of the humanacceptor framework.

As a result, the modified framework 1.4 (hereinafter referred to asrFW1.4) is compatible with virtually any rabbit CDRs. Moreover, rFW1.4containing different rabbit CDRs is well expressed and good producedcontrary to the rabbit wild type single chains and still almost fullyretains the affinity of the original donor rabbit antibodies.

Thus, the present invention provides the variable heavy chain frameworkof SEQ ID No. 1, further comprising one or more amino acid residues thatgenerally support conformation of CDRs derived from a rabbitimmunobinder. In particular, said residues are present at one or moreamino acid positions selected from the group consisting of 24H, 25H,56H, 82H, 84H, 89H and 108H (AHo numbering). These positions are proveto affect CDR conformation and are therefore contemplated for mutationto accommodate donor CDRs. Preferably, said one or more residues areselected from the group consisting of: Threonine (T) at position 24,Valine (V) at position 25, Glycine or Alanine (G or A) at position 56,Lysine (K) at position 82, Threonine (T) at position 84, Valine (V) atposition 89 and Arginine (R) at position 108 (AHo numbering).Preferably, at least three, more preferably, four, five, six and mostpreferably all seven residues are present. Surprisingly, it has beenfound that the presence of the mentioned residues improves the stabilityof the immunobinder.

In a preferred embodiment, the invention provides an immunobinderacceptor framework comprising a VH having at least 50%, more preferablyat least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and even morepreferably 100% identity to SEQ ID No. 4, with the provisio that atleast one, more preferably at least three, more preferably, four, five,six and most preferably seven residues of the group consisting ofthreonine (T) at position 24, valine (V) at position 25, alanine (A) orglycine (G) at position 56, threonine (T) at position 84, lysine (K) atposition 82, valine (V) at position 89 and arginine (R) at position 108(AHo numbering) are present. In a preferred embodiment, the immunobinderacceptor framework is an immunobinder acceptor framework for rabbitCDRs.

In a preferred embodiment, said variable heavy chain framework is orcomprises SEQ ID No. 4 or SEQ ID No. 6. Both of said variable heavychain frameworks may for example be combined with any suitable lightchain framework.

Accordingly, the present invention provides an immunobinder acceptorframework comprising

(i) a variable heavy chain framework having at least 70% identity,preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%identity, to SEQ ID No. 4; and/or

(ii) a variable light chain framework having at least 70% identity,preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%identity, to SEQ ID No. 2.

In a much preferred embodiment, the variable heavy chain frameworkcomprises threonine (T) at position 24, glycine (G) at position 56,threonine (T) at position 84, valine (V) at position 89 and arginine (R)at position 108 (AHo numbering).

In a preferred embodiment, the variable light chain comprises Threonine(T) at position 87 (AHo numbering).

In a preferred embodiment, said immunobinder acceptor frameworkcomprises

(i) a variable heavy chain framework selected from the group consistingof SEQ ID No. 1, SEQ ID No. 4 and SEQ ID No. 6; and/or

(ii) a variable light chain framework of SEQ ID No. 2 or SEQ ID No. 9.

In a preferred embodiment, the variable heavy chain framework is linkedto a variable light chain framework via a linker. The linker may be anysuitable linker, for example a linker comprising 1 to 4 repeats of thesequence GGGGS (SEQ ID NO: 26), preferably a (GGGGS)₄ peptide (SEQ IDNo. 8), or a linker as disclosed in Alfthan et al. (1995) Protein Eng.8:725-731.

In another preferred embodiment, the immunobinder acceptor framework isa sequence having at least 70%, 75%, 80%, 85%, 90% more preferably atleast 95% identity, to SEQ ID No. 5, whereas the sequence, preferably,is not SEQ ID No. 3. More preferably, the immunobinder acceptorframework comprises or is SEQ ID No. 5.

In another preferred embodiment, the immunobinder acceptor framework isa sequence having at least 70%, 75%, 80%, 85%, 90%, more preferably atleast 95% identity, to SEQ ID No. 7, whereas the sequence, preferably,is not SEQ ID No. 3. More preferably, the immunobinder acceptorframework comprises or is SEQ ID No. 7.

Moreover, it was surprisingly found that the presence of the abovedescribed amino acid motif renders a framework, preferably a humanframework, particularly suitable for the accommodation of CDRs fromother non-human animal species, in particular rabbit CDRs. Said motifhas no negative impact on the stability of an immunobinder. The CDRs arepresented in a conformation similar to their native spatial orientationin the rabbit immunobinder; thus, no structurally relevant positionsneed to be grafted onto the acceptor framework. Accordingly, the humanor humanized immunobinder acceptor framework comprises at least threeamino acids, preferably four, five, six and more preferably seven aminoacids of the group consisting of threonine (T) at position 24, valine(V) at position 25, alanine (A) or glycine (G) at position 56, lysine(K) at position 82, threonine (T) at position 84, valine (V) at position89 and arginine (R) at position 108 (AHo numbering).

The immunobinder acceptor frameworks as described herein may comprisesolubility enhancing substitution in the heavy chain framework,preferably at positions 12, 103 and 144 (AHo numbering). Preferably, ahydrophobic amino acid is substituted by a more hydrophilic amino acid.Hydrophilic amino acids are e.g. Arginine (R), Asparagine (N), Asparticacid (D), Glutamine (Q), Glycine (G), Histidine (H), Lysine (K), Serine(S) and Threonine (T). More preferably, the heavy chain frameworkcomprises (a) Serine (S) at position 12; (b) Serine (S) or Threonine (T)at position 103 and/or (c) Serine (S) or Threonine (T) at position 144.

Moreover, stability enhancing amino acids may be present at one or morepositions 1, 3, 4, 10, 47, 57, 91 and 103 of the variable light chainframework (AHo numbering). More preferably, the variable light chainframework comprises glutamic acid (E) at position 1, valine (V) atposition 3, leucine (L) at position 4, Serine (S) at position 10;Arginine (R) at position 47, Serine (S) at position 57, phenylalanine(F) at position 91 and/or Valine (V) at position 103.

As glutamine (Q) is prone to desamination, in another preferredembodiment, the VH comprises at position 141 a glycine (G). Thissubstitution may improve long-term storage of the protein.

For example, the acceptor frameworks disclosed herein can be used togenerate a human or humanized antibody which retains the bindingproperties of the non-human antibody from which the non-human CDRs arederived. Accordingly, in a preferred embodiment the inventionencompasses an immunobinder acceptor framework as disclosed herein,further comprising heavy chain CDR1, CDR2 and CDR3 and/or light chainCDR1, CDR2 and CDR3 from a donor immunobinder, preferably from amammalian immunobinder, more preferably from a lagomorph immunobinderand most preferably from a rabbit. Thus, in one embodiment, theinvention provides an immunobinder specific to a desired antigencomprising

(i) variable light chain CDRs of a lagomorph; and

(ii) a human variable heavy chain framework having at least 50% identityto SEQ ID NO. 4.

In one preferred embodiment, there is the provisio that at least oneamino acid of the group consisting of threonine (T) at position 24,valine (V) at position 25, alanine (A) or glycine (G) at position 56,threonine (T) at position 84, lysine (K) at position 82, valine (V) atposition 89 and arginine (R) at position 108 (AHo numbering) is presentin said human variable heavy chain framework sequence.

Preferably, the lagomorph is a rabbit. More preferably, the immunobindercomprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 andCDR3 from the donor immunobinder.

As known in the art, many rabbit VH chains have extra paired cysteinesrelative to the murine and human counterparts. In addition to theconserved disulfide bridge formed between cys22 and cys92, there is alsoa cys21-cys79 bridge as well as an interCDR S—S bridge formed betweenthe last residue of CDRH1 and the first residue of CDR H2 in some rabbitchains. Besides, pairs of cysteine residues in the CDR-L3 are oftenfound. Besides, many rabbit antibody CDRs do not belong to anypreviously known canonical structure. In particular the CDR-L3 is oftenmuch longer than the CDR-L3 of a human or murine counterpart.

As stated before, the grafting of the non-human CDRs onto the frameworksdisclosed herein yields a molecule wherein the CDRs are displayed in aproper conformation. If required, the affinity of the immunobinder maybe improved by grafting antigen interacting framework residues of thenon-human donor immunobinder. These positions may e.g. be identified by

(i) identifying the respective germ line progenitor sequence or,alternatively, by using the consensus sequences in case of highlyhomologous framework sequences;

(ii) generating a sequence alignment of donor variable domain sequenceswith germ line progenitor sequence or consensus sequence of step (i);and

(iii) identifying differing residues.

Differing residues on the surface of the molecule were in many casesmutated during the affinity generation process in vivo, presumably togenerate affinity to the antigen.

In another aspect, the present invention provides an immunobinder whichcomprises the immunobinder acceptor framework described herein. Saidimmunobinder may e.g. be a scFv antibody, a full-length immunoglobulin,a Fab fragment, a Dab or a Nanobody.

In a preferred embodiment, the immunobinder is attached to one or moremolecules, for example a therapeutic agent such as a cytotoxic agent, acytokine, a chemokine, a growth factor or other signaling molecule, animaging agent or a second protein such as a transcriptional activator ora DNA-binding domain.

The immunobinder as disclosed herein may e.g. be used in diagnosticapplications, therapeutic application, target validation or genetherapy.

The invention further provides an isolated nucleic acid encoding theimmunobinder acceptor framework disclosed herein or the immunobinder(s)as disclosed herein.

In another embodiment, a vector is provided which comprises the nucleicacid disclosed herein.

The nucleic acid or the vector as disclosed herein can e.g. be used ingene therapy.

The invention further encompasses a host cell comprising the vectorand/or the nucleic acid disclosed herein.

Moreover, a composition is provided, comprising the immunobinderacceptor framework as disclosed herein, the immunobinder as disclosedherein, the isolated nucleic acid as disclosed herein or the vector asdisclosed herein.

The sequences disclosed herein are the following (X residues are CDRinsertion sites):

SEQ ID NO.1: variable heavy chain framework of  FW1.4 (a43)EVQLVESGGGLVQPGGSLRLSCAAS(X)_(n=1-50)  WVRQAPGKGLEWVS(X)_(n=1-50) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK(X)_(n=1-50)  WGQGTL VTVSSSEQ ID NO. 2: variable light chain framework of  FW1.4 (KI27)EIVMTQSPSTLSASVGDRVIITC(X)_(n=1-50)  WYQQKPGKAPKLLIY(X)_(n=1-50) GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)_(n=1-50)  FGQGTKLT VLGSEQ ID NO. 3: framework of FW1.4 EIVMTQSPSTLSASVGDRVIITC(X)_(n=1-50) WYQQKPGKAPKLLIY(X)_(n=1-50) GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)_(n=1-50) FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGS  EVQLVESGGGLVQPGGSLRLSCAAS(X)_(n=1-50) WVRQAPGKGLEWVS(X)_(n=1-50) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK(X)_(n=1-50) WGQGTLVTVSSSEQ ID NO. 4: variable heavy chain framework of  rFW1.4EVQLVESGGGLVQPGGSLRLSCTAS(X)_(n=1-50)  WVRQAPGKGLEWVG(X)_(n=1-50)RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR(X)_(n=1-50)  WGQGTLV TVSSSEQ ID NO. 5: framework of rFW1.4 EIVMTQSPSTLSASVGDRVIITC(X)_(n=1-50) WYQQKPGKAPKLLIY(X)_(n=1-50)GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)_(n=1-50) FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTAS(X)_(n=1-50) WVRQAPGKGLEWVG(X)_(n=1-50) RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR(X)_(n=1-50)  WGQGTLVTVSSSEQ ID NO. 6: variable heavy chain framework of  rFW1.4(V2)EVQLVESGGGLVQPGGSLRLSCTVS(X)_(n=1-50)  WVRQAPGKGLEWVG(X)_(n=1-50)RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR(X)_(n=1-50)  WGQGTLVTVSSSEQ ID NO. 7: framework of rFW1.4(V2)EIVMTQSPSTLSASVGDRVIITC(X)_(n=1-50)  WYQQKPGKAPKLLIY(X)_(n=1-50)GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)_(n=1-50) FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGS  EVQLVESGGGLVQPGGSLRLSCTVS(X)_(n=1-50)WVRQAPGKGLEWVG(X)_(n=1-50) RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR(X)_(n=1-50) WGQGTLVTVSS SEQ ID NO. 8: linkerGGGGSGGGGSGGGGSGGGGS SEQ ID NO. 9: substituted variable light chain framework of FW1.4 EIVMTQSPSTLSASVGDRVIITC(X)_(n=1-50) WYQQKPGKAPKLLIY(X)_(n=1-50)GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)_(n=1-50)  FGQGTKLTVLG

In another aspect, the invention provides methods for the humanizationof non-human antibodies by grafting CDRs of non-human donor antibodiesonto stable and soluble antibody frameworks. In a particularly preferredembodiment, the CDRs stem from rabbit antibodies and the frameworks arethose described above.

A general method for grafting CDRs into human acceptor frameworks hasbeen disclosed by Winter in U.S. Pat. No. 5,225,539 and by Queen et al.in WO9007861A1, which are hereby incorporated by reference in theirentirety. The general strategy for grafting CDRs from rabbit monoclonalantibodies onto selected frameworks is related to that of Winter et al.and Queen et al., but diverges in certain key respects. In particular,the methods of the invention diverge from the typical Winter and Queenmethodology known in the art in that the human antibody frameworks asdisclosed herein are particularly suitable as universal acceptors forhuman or non-human donor antibodies. Thus, unlike the general method ofWinter and Queen, the framework sequence used for the humanizationmethods of the invention is not necessarily the framework sequence whichexhibits the greatest sequence similarity to the sequence of thenon-human (e.g., rabbit) antibody from which the donor CDRs are derived.In addition, framework residue grafting from the donor sequence tosupport CDR conformation is not required. At most, antigen binding aminoacids located in the framework or other mutations that occurred duringsomatic hypermutation may be introduced.

Particular details of the grafting methods to generate humanizedrabbit-derived antibodies with high solubility and stability aredescribed below.

Accordingly, the invention provides a method of humanizing a rabbit CDRdonor immunobinder which comprises heavy chain CDR1, CDR2 and CDR3sequences and/or light chain CDR1, CDR2 and CDR3 sequences. The methodcomprises the steps of:

(i) grafting onto the heavy chain at least one, preferably two, morepreferably three CDRs of the group consisting of CDR1, CDR2 and CDR3sequences into a human heavy chain acceptor framework having at least50%, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, morepreferably at least 95% identity to SEQ ID NO:1; and/or

(ii) grafting onto the light chain at least one, preferably two, morepreferably three CDRs of the group consisting of CDR1, CDR2 and CDR3sequences into a human light chain acceptor framework, the human lightchain framework having at least 50%, preferably at least 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, more preferably at least 95% identity to SEQ IDNO:2.

In a preferred embodiment, the variable chain acceptor frameworkcomprises (i) a human heavy chain framework comprising a framework aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:4 and SEQ ID NO:6 and (ii) a human light chain framework comprisingthe framework amino acid sequence of SEQ ID NO:2 or SEQ ID NO:9.

In a much preferred embodiment, the method comprises the step of (i)grafting the heavy chain CDR1, CDR2 and CDR3 sequences into the heavychain and (ii) grafting the light chain CDR1, CDR2 and CDR3 sequencesinto the light chain of an immunobinder having at least 75%, 80%, 85%,90%, more preferably at least 95% identity to SEQ ID No. 3, SEQ ID No. 5or SEQ ID No. 7. More preferably, the immunobinder is or comprises SEQID No. 3, SEQ ID No. 5 or SEQ ID No. 7.

In another embodiment, in order to improve antigen binding, the methodmay further comprise the step of substituting acceptor frameworkresidues by donor residues which are involved in antigen binding.

In exemplary embodiments of the methods of the invention, the amino acidsequence of the CDR donor antibody is first identified and the sequencesaligned using conventional sequence alignment tools (e.g.,Needleman-Wunsch algorithm and Blossum matrices). The introduction ofgaps and nomenclature of residue positions may be done using aconventional antibody numbering system. For example, the AHo numberingsystem for immunoglobulin variable domains may be used. The Kabatnumbering scheme may also be applied since it is the most widely adoptedstandard for numbering the residues in an antibody. Kabat numbering maye.g. be assigned using the SUBIM program. This program analyses variableregions of an antibody sequence and numbers the sequence according tothe system established by Kabat and co-workers (Deret et al 1995). Thedefinition of framework and CDR regions is generally done following theKabat definition which is based on sequence variability and is the mostcommonly used. However, for CDR-H1, the designation is preferably acombination of the definitions of Kabat's, mean contact data generatedby analysis of contacts between antibody and antigen of a subset of 3Dcomplex structures (MacCallum et al., 1996) and Chotia's which is basedon the location of the structural loop regions (see also FIG. 1).Conversion tables for the two different numbering systems used toidentify amino acid residue positions in antibody heavy and light chainvariable regions are provided in A. Honegger, J. Mol. Biol. 309 (2001)657-670. The Kabat numbering system is described further in Kabat et al.(Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). The AHo numbering system is describedfurther in Honegger, A. and Pluckthun, A. (2001) J Mol. Biol.309:657-670).

The variable domains of the rabbit monoclonal antibodies may e.g. beclassified into corresponding human sub-groups using e.g. an EXCELimplementation of sequence analysis algorithms and classificationmethods based on analysis of the human antibody repertoire (Knappik etal., 2000, J Mol Biol. February 11; 296(1):57-86).

CDR conformations may be assigned to the donor antigen binding regions,subsequently residue positions required to maintain the differentcanonical structures can also be identified. The CDR canonicalstructures for five of the six antibody hypervariable regions of rabbitantibodies (L1, L2, L3, H1 and H2) are determined using Chothia's (1989)definition.

In a preferred embodiment, the CDRs are generated, identified andisolated according to the following method: B-cells, preferably rabbitB-cells, are incubated with (i) target antigens (preferably purified) or(ii) with cells expressing the target antigen on their surface.

In case (ii), said cells expressing the target antigen may e.g. bemammalian cells, preferably CHO or HEK293 cells, yeast cells, preferablyyeast spheroblasts, or bacterial cells which naturally express thetarget of choice or are transformed to express the target protein ontheir surface. Upon expression, the target antigen may be expressed onthe cell surface either integrated or attached to the cell membrane. Thecells may e.g. be cultivated as isolated strains in cell culture or beisolated from their natural environment, e.g. a tissue, an organ or anorganism.

Providing the target antigen expressed on the surface of cells, i.e.case (ii), is especially preferred for transmembrane proteins, even morepreferably for multi-membrane-spanning proteins, such as GPCRs (Gprotein-coupled receptors) or ion channels or any other protein of whichthe native conformation is difficult to maintain upon recombinantexpression and purification. Traditional immunization with therecombinant protein is in these cases inadvisable or impossible due toloss of native conformation of integral membrane proteins/complexesduring the purification process or due to insufficient amounts of pureprotein. In a preferred embodiment of the invention, a mammal, morepreferably a rabbit, is immunized with DNA instead of recombinantprotein e.g. by a DNA vaccination protocol as disclosed inWO/2004/087216. DNA vaccination induces a rapid immune response againsta native antigen. Since no recombinant protein is needed, thistechnology is on one hand very cost-effective, on the other hand, andmore importantly, this method allows for native expression of integralmembrane complexes and/or multi-membrane-spanning membrane proteins. TheB-cells may be isolated from said immunized mammal, preferably of saidrabbit, or alternatively be naive B-cells.

In a subsequent step of said method B-cells, preferably memory B-cells,are isolated from lymphatic organs of the immunized animal (such asspleen or lymph nodes), preferably of immunized rabbits. The B-cells areincubated in a mixture with either cells expressing the antigen on theirsurface or with fluorescence-labeled soluble antigen. B-cells thatexpress target specific antibodies on their surface and consequentlybind to the target antigen or to the target antigen expressed on thecell surface are isolated. In a much preferred embodiment, the B-cellsand/or the target cells are stained to allow for isolation via flowcytometry based sorting of B-cell/target cell or B-cell/antigencomplexes. Flow cytometry normally measures the fluorescence emitted bysingle cells when they cross a laser beam. However, some researchershave already used cytometers to investigate cell-cell interactions, forexample adhesion mediated by cadherins (Panorchan et al, 2006, J. CellScience, 119, 66-74; Leong et Hibma, 2005, J. Immunol. Methods, 302,116-124) or integrins (Gawaz et al, 1991, J. Clin. Invest, 88,1128-1134). Thus, in case (ii), cells expressing the target of choiceare stained with an intracellular fluorescent dye (for example calcein).B-cells are stained with fluorescent antibodies binding to cell surfacespecific markers. Thus, bi-color “events” may be selected, consisting intwo cells adhering to each other through B-cell receptor-targetinteractions (see FIG. 2).

As IgG have generally a higher affinity as IgMs, preferably, positiveB-cells expressing IgG but not IgM on their surface (which ischaracteristic for memory B-cells) are selected. For said purpose,multicolor staining is preferably used, where antibodies specific forIgG and IgM are differentially labeled, e.g. with APC and FITC,respectively.

In one particular embodiment, a read-out for B-cell sorting can alsoselect for the ability of this interaction to functionallyblock/activate receptor signaling. For example, B-cells could beincubated with cells that functionally express a GPCR (G protein-coupledreceptor). An agonist that signals through a GPCR can be added to themixture to induce GPCR mediated Ca2+ efflux from the endoplasmicreticulum. In case an antibody presented on a B-cell would functionallyblock agonist signaling, Ca2+ efflux would consequently also be blockedby this cell-cell interaction. Ca2+ efflux can be quantitativelymeasured by flow-cytometry. Therefore, only B-cell/target cellconglomerates that either show increase or decrease in Ca2+ efflux wouldbe sorted.

The selection step is followed by the cultivation of the B-cells undersuitable conditions so that antibodies are secreted into the culturemedium. The produced antibodies are monoclonal antibodies. Thecultivation may involve the use of a helper cell line, such as a thymomahelper cell line (e.g. EL4-B5, see Zubler et al, 1985, J. Immunol.,134(6): 3662-3668). Preferably, a validation step is performed testingthe generated antibodies for specific binding to the target, e.g. forexcluding antibodies which are directed against a protein beingexpressed on the cell surface other than the target protein. Forexample, CELISA, i.e. a modified enzyme-linked immunosorbent assay(ELISA), where coating step is performed with entire cells, is suitablefor said purpose. Said method allows for the evaluation of theselectivity and the ability of antibodies to compete with the ligand.

The antibodies generated in the above mentioned step are then analyzedto identify the CDRs of said antibodies. This may involve steps such aspurifying the antibodies, elucidating their amino acid sequence and/ornucleic acid sequence.

Finally, the CDRs may then be grafted onto acceptor frameworks e.g. bygene synthesis with the oligo extension method, preferably onto theacceptor frameworks described above. In one embodiment, the artrecognized process of CDR grafting can be used to transfer donor CDRsinto acceptor frameworks. In most cases, all three CDRs from the heavychain are transplanted from the donor antibody to a single acceptorframework and all three CDRs from the light chain are transplanted to adifferent acceptor framework. It is expected that it should not alwaysbe necessary to transplant all the CDRs, as some CDRs may not beinvolved in binding to antigen, and CDRs with different sequences (andthe same length) can have the same folding (and therefore contacts fromantigen to the main chain contacts could be retained despite thedifferent sequences). Indeed single domains (Ward et al, 1989, Nature341, pp. 544-546) or even single CDRs (R. Taub et al, 1989, J. Biol Chem264, pp. 259-265) can have antigen binding activities alone. However,whether all or only some of the CDRs are transplanted, the intention ofCDR grafting is to transplant the same, or much the same antigen bindingsite, from animal to human antibodies (see, e.g., U.S. Pat. No.5,225,539 (Winter)).

In another embodiment, the CDRs of the donor antibody can be alteredprior to or after their incorporation into the acceptor framework.

Alternatively, characterization of the antibodies would be performedonly in their final immunobinder format. For this approach, CDRsequences of antibodies expressed on sorted B-cells are retrieved byRT-PCR from either the cultured sorted B-cells or from single sortedB-cells directly. For said purpose, mulitplication of B-cells and/or thevalidation step described above and/or the analyzation step as describedabove may not be necessary. Combination of two pools of partiallyoverlapping oligonucleotides in which one oligonucleotide pool is codingfor the CDRs and a second pool encodes the framework regions of asuitable immunobinder scaffold would allow to generate a humanizedimmunobinders in a one-step PCR procedure. Highthroughput sequencing,cloning and production would allow to perform clone selection based onthe performance of the purified humanized immunobinders, instead ofcharacterizing secreted IgG in the cell culture supernatant. In apreferred embodiment thereof, the immunobinder is a scFv.

However, grafting of CDRs may result in an impaired affinity of thegenerated immunobinder to the antigen due to framework residues whichare in contact with the antigen. Such interactions may be a result ofsomatic hypermutation. Therefore, it may still be required to graft suchdonor framework amino acids onto the framework of the humanizedantibody. Amino acid residues from the non-human immunobinder involvedin antigen binding may be identified by examination of rabbit monoclonalantibody variable region sequences and structures. Each residue in theCDR donor framework that differs from the germline may be considered asrelevant. If the closest germline cannot be established, the sequencecan be compared against the subgroup consensus or the consensus ofrabbit sequences with a high percentage of similarity. Rare frameworkresidues are considered as possible result of somatic hypermutation andtherefore as playing a role in binding.

The antibodies of the invention may be further optimized to showenhanced functional properties, e.g., enhanced solubility and/orstability. In certain embodiments, the antibodies of the invention areoptimized according to the “functional consensus” methodology disclosedin PCT Application Serial No. PCT/EP2008/001958, entitled “SequenceBased Engineering and Optimization of Single Chain Antibodies”, filed onMar. 12, 2008, which is incorporated herein by reference.

For example, the immunobinders of the invention can be compared with adatabase of functionally-selected scFvs is used to identify amino acidresidue positions that are either more or less tolerant of variabilitythan the corresponding positions in immunobinder, thereby indicatingthat such identified residue positions may be suitable for engineeringto improve functionality such as stability and/or solubility.

Exemplary framework residue positions for substitution and exemplaryframework substitutions are described in PCT Application No.PCT/CH2008/000285, entitled “Methods of Modifying Antibodies, andModified Antibodies with Improved Functional Properties”, filed on Jun.25, 2008, and PCT Application No. PCT/CH2008/000284, entitled “SequenceBased Engineering and Optimization of Single Chain Antibodies”, filed onJun. 25, 2008. For example, one or more of the following substitutionsmay be introduced at an amino acid position (AHo numbering is referencedfor each of the amino acid position listed below) in the heavy chainvariable region of an immunobinder of the invention:

-   -   (a) Q or E at amino acid position 1;    -   (b) Q or E at amino acid position 6;    -   (c) T, S or A at amino acid position 7, more preferably T or A,        even more preferably T;    -   (d) A, T, P, V or D, more preferably T, P, V or D, at amino acid        position 10,    -   (e) L or V, more preferably L, at amino acid position 12,    -   (f) V, R, Q, M or K, more preferably V, R, Q or M at amino acid        position 13;    -   (g) R, M, E, Q or K, more preferably R, M, E or Q, even more        preferably R or E, at amino acid position 14;    -   (h) L or V, more preferably L, at amino acid position 19;    -   (i) R, T, K or N, more preferably R, T or N, even more        preferably N, at amino acid position 20;    -   (j) I, F, L or V, more preferably I, F or L, even more        preferably I or L, at amino acid position 21;    -   (k) R or K, more preferably K, at amino acid position 45;    -   (l) T, P, V, A or R, more preferably T, P, V or R, even more        preferably R, at amino acid position 47;    -   (m) K, Q, H or E, more preferably K, H or E, even more        preferably K, at amino acid position 50;    -   (n) M or I, more preferably I, at amino acid position 55;    -   (o) K or R, more preferably K, at amino acid position 77;    -   (p) A, V, L or I, more preferably A, L or I, even more        preferably A, at amino acid position 78;    -   (q) E, R, T or A, more preferably E, T or A, even more        preferably E, at amino acid position 82;    -   (r) T, S, I or L, more preferably T, S or L, even more        preferably T, at amino acid position 86;    -   (s) D, S, N or G, more preferably D, N or G, even more        preferably N, at amino acid position 87;    -   (t) A, V, L or F, more preferably A, V or F, even more        preferably V, at amino acid position 89;    -   (u) F, S, H, D or Y, more preferably F, S, H or D, at amino acid        position 90;    -   (v) D, Q or E, more preferably D or Q, even more preferably D,        at amino acid position 92;    -   (w) G, N, T or S, more preferably G, N or T, even more        preferably G, at amino acid position 95;    -   (x) T, A, P, F or S, more preferably T, A, P or F, even more        preferably F, at amino acid position 98;    -   (y) R, Q, V, I, M, F, or L, more preferably R, Q, I, M, F or L,        even more preferably Y, even more preferably L, at amino acid        position 103; and    -   (z) N, S or A, more preferably N or S, even more preferably N,        at amino acid position 107.

Additionally or alternatively, one or more of the followingsubstitutions can be introduced into the light chain variable region ofan immunobinder of the invention:

-   -   (aa) Q, D, L, E, S, or I, more preferably L, E, S or I, even        more preferably L or E, at amino acid position 1;    -   (bb) S, A, Y, I, P or T, more preferably A, Y, I, P or T, even        more preferably P or T at amino acid position 2;    -   (cc) Q, V, T or I, more preferably V, T or I, even more        preferably V or T, at amino acid position 3;    -   (dd) V, L, I or M, more preferably V or L, at amino acid        position 4;    -   (ee) S, E or P, more preferably S or E, even more preferably S,        at amino acid position 7;    -   (ff) T or I, more preferably I, at amino acid position 10;    -   (gg) A or V, more preferably A, at amino acid position 11;    -   (hh) S or Y, more preferably Y, at amino acid position 12;    -   (ii) T, S or A, more preferably T or S, even more preferably T,        at amino acid position 14;    -   (jj) S or R, more preferably S, at amino acid position 18;    -   (kk) T or R, more preferably R, at amino acid position 20;    -   (ll) R or Q, more preferably Q, at amino acid position 24;    -   (mm) H or Q, more preferably H, at amino acid position 46;    -   (nn) K, R or I, more preferably R or I, even more preferably R,        at amino acid position 47;    -   (oo) R, Q, K, E, T, or M, more preferably Q, K, E, T or M, at        amino acid position 50;    -   (pp) K, T, S, N, Q or P, more preferably T, S, N, Q or P, at        amino acid position 53;    -   (qq) I or M, more preferably M, at amino acid position 56;    -   (rr) H, S, F or Y, more preferably H, S or F, at amino acid        position 57;    -   (ss) I, V or T, more preferably V or T, R, even more preferably        T, at amino acid position 74;    -   (tt) R, Q or K, more preferably R or Q, even more preferably R,        at amino acid position 82;    -   (uu) L or F, more preferably F, at amino acid position 91;    -   (vv) G, D, T or A, more preferably G, D or T, even more        preferably T, at amino acid position 92;    -   (xx) S or N, more preferably N, at amino acid position 94;    -   (yy) F, Y or S, more preferably Y or S, even more preferably S,        at amino acid position 101; and    -   (zz) D, F, H, E, L, A, T, V, S, G or I, more preferably H, E, L,        A, T, V, S, G or I, even more preferably A or V, at amino acid        position 103.

In other embodiments, the immunobinders of the invention comprise one ormore of the stability enhancing mutations described in U.S. ProvisionalApplication Ser. No. 61/075,692, entitled “Solubility Optimization ofImmunobinders”, filed on Jun. 25, 2008. In certain preferredembodiments, the immunobinder comprises a solubility enhancing mutationat an amino acid position selected from the group of heavy chain aminoacid positions consisting of 12, 103 and 144 (AHo Numbering convention).In one preferred embodiment, the immunobinder comprises one or moresubstitutions selected from the group consisting of: (a) Serine (S) atheavy chain amino acid position 12; (b) Serine (S) or Threonine (T) atheavy chain amino acid position 103; and (c) Serine (S) or Threonine (T)at heavy chain amino acid position 144. In another embodiment, theimmunobinder comprises the following substitutions: (a) Serine (S) atheavy chain amino acid position 12; (b) Serine (S) or Threonine (T) atheavy chain amino acid position 103; and (c) Serine (S) or Threonine (T)at heavy chain amino acid position 144.

In certain preferred embodiments, the immunobinder comprises stabilityenhancing mutations at a framework residue of the light chain acceptorframework in at least one of positions 1, 3, 4, 10, 47, 57, 91 and 103of the light chain variable region according to the AHo numberingsystem. In a preferred embodiment, the light chain acceptor frameworkcomprises one or more substitutions selected from the group consistingof (a) glutamic acid (E) at position 1, (b) valine (V) at position 3,(c) leucine (L) at position 4; (d) Serine (S) at position 10; (e)Arginine (R) at position 47; (e) Serine (S) at position 57; (f)phenylalanine (F) at position 91; and (g) Valine (V) at position 103.

One can use any of a variety of available methods to produce a humanizedantibody comprising a mutation as described above.

Accordingly, the present invention provides an immunobinder humanizedaccording to the method described herein.

In certain preferred embodiments, the target antigen of saidimmunobinder is VEGF or TNFα.

The polypeptides described in the present invention or generated by amethod of the present invention can, for example, be synthesized usingtechniques known in the art. Alternatively nucleic acid moleculesencoding the desired variable regions can be synthesized and thepolypeptides produced by recombinant methods.

For example, once the sequence of a humanized variable region has beendecided upon, that variable region or a polypeptide comprising it can bemade by techniques well known in the art of molecular biology. Morespecifically, recombinant DNA techniques can be used to produce a widerange of polypeptides by transforming a host cell with a nucleic acidsequence (e.g., a DNA sequence that encodes the desired variable region(e.g., a modified heavy or light chain; the variable domains thereof, orother antigen-binding fragments thereof)).

In one embodiment, one can prepare an expression vector including apromoter that is operably linked to a DNA sequence that encodes at leastV_(H) or V_(L). If necessary, or desired, one can prepare a secondexpression vector including a promoter that is operably linked to a DNAsequence that encodes the complementary variable domain (i.e., where theparent expression vector encodes V_(H), the second expression vectorencodes V_(L) and vice versa). A cell line (e.g., an immortalizedmammalian cell line) can then be transformed with one or both of theexpression vectors and cultured under conditions that permit expressionof the chimeric variable domain or chimeric antibody (see, e.g.,International Patent Application No. PCT/GB85/00392 to Neuberger et.al.).

In one embodiment, variable regions comprising donor CDRs and acceptorFR amino acid sequences can be made and then changes introduced into thenucleic acid molecules to effect the CDR amino acid substitution.

Exemplary art recognized methods for making a nucleic acid moleculeencoding an amino acid sequence variant of a polypeptide include, butare not limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the polypeptide.

Site-directed mutagenesis is a preferred method for preparingsubstitution variants. This technique is well known in the art (see,e.g., Carter et al. Nucleic Acids Res. 13:4431-4443 (1985) and Kunkel etal., Proc. Natl. Acad. Sci. USA 82:488 (1987)). Briefly, in carrying outsite-directed mutagenesis of DNA, the parent DNA is altered by firsthybridizing an oligonucleotide encoding the desired mutation to a singlestrand of such parent DNA. After hybridization, a DNA polymerase is usedto synthesize an entire second strand, using the hybridizedoligonucleotide as a primer, and using the single strand of the parentDNA as a template. Thus, the oligonucleotide encoding the desiredmutation is incorporated in the resulting double-stranded DNA.

PCR mutagenesis is also suitable for making amino acid sequence variantsof polypeptides. See Higuchi, in PCR Protocols, pp. 177-183 (AcademicPress, 1990); and Vallette et al., Nuc. Acids Res. 17:723-733 (1989).Briefly, when small amounts of template DNA are used as startingmaterial in a PCR, primers that differ slightly in sequence from thecorresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al., Gene 34:315-323 (1985). Thestarting material is the plasmid (or other vector) comprising the DNA tobe mutated. The codon(s) in the parent DNA to be mutated are identified.There must be a unique restriction endonuclease site on each side of theidentified mutation site(s). If no such restriction sites exist, theymay be generated using the above-described oligonucleotide-mediatedmutagenesis method to introduce them at appropriate locations in the DNAencoding the polypeptide. The plasmid DNA is cut at these sites tolinearize it. A double-stranded oligonucleotide encoding the sequence ofthe DNA between the restriction sites but containing the desiredmutation(s) is synthesized using standard procedures, wherein the twostrands of the oligonucleotide are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 5′ and 3′ ends that are compatible with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated DNA sequence.

A variable region generated by the methods of the invention can bere-modeled and further altered to further increase antigen binding.Thus, the steps described above can be preceded or followed byadditional steps, including, e.g. affinity maturation. In addition,empirical binding data can be used for further optimization.

It will be understood by one of ordinary skill in the art that thepolypeptides of the invention may further be modified such that theyvary in amino acid sequence, but not in desired activity. For example,additional nucleotide substitutions leading to amino acid substitutionsat “non-essential” amino acid residues may be made to the protein Forexample, a nonessential amino acid residue in an immunoglobulinpolypeptide may be replaced with another amino acid residue from thesame side chain family. In another embodiment, a string of amino acidscan be replaced with a structurally similar string that differs in orderand/or composition of side chain family members, i.e., a conservativesubstitution, in which an amino acid residue is replaced with an aminoacid residue having a similar side chain, may be made.

Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Aside from amino acid substitutions, the present invention contemplatesother modifications, e.g., to Fc region amino acid sequences in order togenerate an Fc region variant with altered effector function. One may,for example, delete one or more amino acid residues of the Fc region inorder to reduce or enhance binding to an FcR. In one embodiment, one ormore of the Fc region residues can be modified in order to generate suchan Fc region variant. Generally, no more than one to about ten Fc regionresidues will be deleted according to this embodiment of the invention.The Fc region herein comprising one or more amino acid deletions willpreferably retain at least about 80%, and preferably at least about 90%,and most preferably at least about 95%, of the starting Fc region or ofa native sequence human Fc region.

One may also make amino acid insertion Fc region variants, whichvariants have altered effector function. For example, one may introduceat least one amino acid residue (e.g. one to two amino acid residues andgenerally no more than ten residues) adjacent to one or more of the Fcregion positions identified herein as impacting FcR binding. By“adjacent” is meant within one to two amino acid residues of an Fcregion residue identified herein. Such Fc region variants may displayenhanced or diminished FcRn binding.

Such Fc region variants will generally comprise at least one amino acidmodification in the Fc region. In one embodiment amino acidmodifications may be combined. For example, the variant Fc region mayinclude two, three, four, five, etc substitutions therein, e.g. of thespecific Fc region positions identified herein. In another embodiment, apolypeptide may have altered binding to FcRn and to another Fc receptor.

In one embodiment, the polypeptides described in the present inventionor generated by a method of the present invention, e.g., humanized Igvariable regions and/or polypeptides comprising humanized Ig variableregions may be produced by recombinant methods. For example, apolynucleotide sequence encoding a polypeptide can be inserted in asuitable expression vector for recombinant expression. Where thepolypeptide is an antibody, polynucleotides encoding additional lightand heavy chain variable regions, optionally linked to constant regions,may be inserted into the same or different expression vector. Anaffinity tag sequence (e.g. a His(6) tag) may optionally be attached orincluded within the polypeptide sequence to facilitate downstreampurification. The DNA segments encoding immunoglobulin chains are theoperably linked to control sequences in the expression vector(s) thatensure the expression of immunoglobulin polypeptides. Expression controlsequences include, but are not limited to, promoters (e.g.,naturally-associated or heterologous promoters), signal sequences,enhancer elements, and transcription termination sequences. Preferably,the expression control sequences are eukaryotic promoter systems invectors capable of transforming or transfecting eukaryotic host cells.Once the vector has been incorporated into the appropriate host, thehost is maintained under conditions suitable for high level expressionof the nucleotide sequences, and the collection and purification of thepolypeptide.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362). E. coliis one prokaryotic host particularly useful for cloning thepolynucleotides (e.g., DNA sequences) of the present invention. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilus, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species.

Other microbes, such as yeast, are also useful for expression.Saccharomyces and Pichia are exemplary yeast hosts, with suitablevectors having expression control sequences (e.g., promoters), an originof replication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formethanol, maltose, and galactose utilization.

Within the scope of the present invention, E. coli and S. cerevisiae arepreferred host cells.

In addition to microorganisms, mammalian tissue culture may also be usedto express and produce the polypeptides of the present invention (e.g.,polynucleotides encoding immunoglobulins or fragments thereof). SeeWinnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Eukaryotic cells are actually preferred, because a number of suitablehost cell lines capable of secreting heterologous proteins (e.g., intactimmunoglobulins) have been developed in the art, and include CHO celllines, various Cos cell lines, HeLa cells, 293 cells, myeloma celllines, transformed B-cells, and hybridomas. Expression vectors for thesecells can include expression control sequences, such as an origin ofreplication, a promoter, and an enhancer (Queen et al., Immunol. Rev.89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Coet al., J. Immunol. 148:1149 (1992).

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment, electroporation, lipofection,biolistics or viral-based transfection may be used for other cellularhosts. (See generally Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Press, 2nd ed., 1989). Other methods used totransform mammalian cells include the use of polybrene, protoplastfusion, liposomes, electroporation, and microinjection (see generally,Sambrook et al., supra). For production of transgenic animals,transgenes can be microinjected into fertilized oocytes, or can beincorporated into the genome of embryonic stem cells, and the nuclei ofsuch cells transferred into enucleated oocytes.

The subject polypeptide can also be incorporated in transgenes forintroduction into the genome of a transgenic animal and subsequentexpression, e.g., in the milk of a transgenic animal (see, e.g., Deboeret al. 5,741,957; Rosen 5,304,489; and Meade 5,849,992. Suitabletransgenes include coding sequences for light and/or heavy chains inoperable linkage with a promoter and enhancer from a mammary glandspecific gene, such as casein or beta lactoglobulin.

Polypeptides can be expressed using a single vector or two vectors. Forexample, antibody heavy and light chains may be cloned on separateexpression vectors and co-transfected into cells.

In one embodiment, signal sequences may be used to facilitate expressionof polypeptides of the invention.

Once expressed, the polypeptides can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns (e.g., protein A or protein G), column chromatography,HPLC purification, gel electrophoresis and the like (see generallyScopes, Protein Purification (Springer-Verlag, N.Y., (1982)).

Either the humanized Ig variable regions or polypeptides comprising themcan be expressed by host cells or cell lines in culture. They can alsobe expressed in cells in vivo. The cell line that is transformed (e.g.,transfected) to produce the altered antibody can be an immortalizedmammalian cell line, such as those of lymphoid origin (e.g., a myeloma,hybridoma, trioma or quadroma cell line). The cell line can also includenormal lymphoid cells, such as B-cells, that have been immortalized bytransformation with a virus (e.g., the Epstein-Barr virus).

Although typically the cell line used to produce the polypeptide is amammalian cell line, cell lines from other sources (such as bacteria andyeast) can also be used. In particular, E. coli-derived bacterialstrains can be used, especially, e.g., phage display.

Some immortalized lymphoid cell lines, such as myeloma cell lines, intheir normal state, secrete isolated Ig light or heavy chains. If such acell line is transformed with a vector that expresses an alteredantibody, prepared during the process of the invention, it will not benecessary to carry out the remaining steps of the process, provided thatthe normally secreted chain is complementary to the variable domain ofthe Ig chain encoded by the vector prepared earlier.

If the immortalized cell line does not secrete or does not secrete acomplementary chain, it will be necessary to introduce into the cells avector that encodes the appropriate complementary chain or fragmentthereof.

In the case where the immortalized cell line secretes a complementarylight or heavy chain, the transformed cell line may be produced forexample by transforming a suitable bacterial cell with the vector andthen fusing the bacterial cell with the immortalized cell line (e.g., byspheroplast fusion). Alternatively, the DNA may be directly introducedinto the immortalized cell line by electroporation.

In one embodiment, a humanized Ig variable region as described in thepresent invention or generated by a method of the present invention canbe present in an antigen-binding fragment of any antibody. The fragmentscan be recombinantly produced and engineered, synthesized, or producedby digesting an antibody with a proteolytic enzyme. For example, thefragment can be a Fab fragment; digestion with papain breaks theantibody at the region, before the inter-chain (i.e., V_(H)-V_(H))disulphide bond, that joins the two heavy chains. This results in theformation of two identical fragments that contain the light chain andthe V_(H) and C_(H)1 domains of the heavy chain. Alternatively, thefragment can be an F(ab′)₂ fragment. These fragments can be created bydigesting an antibody with pepsin, which cleaves the heavy chain afterthe inter-chain disulfide bond, and results in a fragment that containsboth antigen-binding sites. Yet another alternative is to use a “singlechain” antibody. Single-chain Fv (scFv) fragments can be constructed ina variety of ways. For example, the C-terminus of V_(H) can be linked tothe N-terminus of V_(L). Typically, a linker (e.g., (GGGGS)₄; SEQ ID NO:8) is placed between V_(H) and V_(L). However, the order in which thechains can be linked can be reversed, and tags that facilitate detectionor purification (e.g., Myc-, His-, or FLAG-tags) can be included (tagssuch as these can be appended to any antibody or antibody fragment ofthe invention; their use is not restricted to scFv). Accordingly, and asnoted below, tagged antibodies are within the scope of the presentinvention. In alternative embodiments, the antibodies described herein,or generated by the methods described herein, can be heavy chain dimersor light chain dimers. Still further, an antibody light or heavy chain,or portions thereof, for example, a single domain antibody (DAb), can beused.

In another embodiment, a humanized Ig variable region as described inthe present invention or generated by a method of the present inventionis present in a single chain antibody (ScFv) or a minibody (see e.g.,U.S. Pat. No. 5,837,821 or WO 94/09817A1). Minibodies are dimericmolecules made up of two polypeptide chains each comprising an ScFvmolecule (a single polypeptide comprising one or more antigen bindingsites, e.g., a V_(L) domain linked by a flexible linker to a V_(H)domain fused to a CH3 domain via a connecting peptide). ScFv moleculescan be constructed in a V_(H)-linker-V_(L) orientation orV_(L)-linker-V_(H) orientation. The flexible hinge that links the V_(L)and V_(H) domains that make up the antigen binding site preferablycomprises from about 10 to about 50 amino acid residues. An exemplaryconnecting peptide for this purpose is (Gly4Ser)3 (SEQ ID NO: 27)(Huston et al. (1988). PNAS, 85:5879). Other connecting peptides areknown in the art.

Methods of making single chain antibodies are well known in the art,e.g., Ho et al. (1989), Gene, 77:51; Bird et al. (1988), Science242:423; Pantoliano et al. (1991), Biochemistry 30:10117; Milenic et al.(1991), Cancer Research, 51:6363; Takkinen et al. (1991), ProteinEngineering 4:837. Minibodies can be made by constructing an ScFvcomponent and connecting peptide-CH₃ component using methods describedin the art (see, e.g., U.S. Pat. No. 5,837,821 or WO 94/09817A1). Thesecomponents can be isolated from separate plasmids as restrictionfragments and then ligated and recloned into an appropriate vector.Appropriate assembly can be verified by restriction digestion and DNAsequence analysis. In one embodiment, a minibody of the inventioncomprises a connecting peptide. In one embodiment, the connectingpeptide comprises a Gly/Ser linker, e.g., GGGSSGGGSGG (SEQ ID NO: 28).

In another embodiment, a tetravalent minibody can be constructed.

Tetravalent minibodies can be constructed in the same manner asminibodies, except that two ScFv molecules are linked using a flexiblelinker, e.g., having an amino acid sequence (G₄S)₄G₃AS (SEQ ID NO: 29).

In another embodiment, a humanized variable region as described in thepresent invention or generated by a method of the present invention canbe present in a diabody. Diabodies are similar to scFv molecules, butusually have a short (less than 10 and preferably 1-5) amino acidresidue linker connecting both variable domains, such that the V_(L) andV_(H) domains on the same polypeptide chain can not interact. Instead,the V_(L) and V_(H) domain of one polypeptide chain interact with theV_(H) and V_(L) domain (respectively) on a second polypeptide chain (WO02/02781).

In another embodiment, a humanized variable region of the invention canbe present in an immunoreactive fragment or portion of an antibody (e.g.an scFv molecule, a minibody, a tetravalent minibody, or a diabody)operably linked to an FcR binding portion. In an exemplary embodiment,the FcR binding portion is a complete Fc region.

Preferably, the humanization methods described herein result in Igvariable regions in which the affinity for antigen is not substantiallychanged compared to the donor antibody.

In one embodiment, polypeptides comprising the variable domains of theinstant invention bind to antigens with a binding affinity greater than(or equal to) an association constant Ka of about 10⁵ M⁻¹, 10⁶ M⁻¹, 10⁷M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, or 10¹² M⁻¹, (includingaffinities intermediate of these values).

Affinity, avidity, and/or specificity can be measured in a variety ofways. Generally, and regardless of the precise manner in which affinityis defined or measured, the methods of the invention improve antibodyaffinity when they generate an antibody that is superior in any aspectof its clinical application to the antibody (or antibodies) from whichit was made (for example, the methods of the invention are consideredeffective or successful when a modified antibody can be administered ata lower dose or less frequently or by a more convenient route ofadministration than an antibody (or antibodies) from which it was made).

More specifically, the affinity between an antibody and an antigen towhich it binds can be measured by various assays, including, e.g., anELISA assay, a BiaCore assay or the KinExA™ 3000 assay (available fromSapidyne Instruments (Boise, Id.)). Briefly, sepharose beads are coatedwith antigen (the antigen used in the methods of the invention can beany antigen of interest (e.g., a cancer antigen; a cell surface proteinor secreted protein; an antigen of a pathogen (e.g., a bacterial orviral antigen (e.g., an HIV antigen, an influenza antigen, or ahepatitis antigen)), or an allergen) by covalent attachment. Dilutionsof antibody to be tested are prepared and each dilution is added to thedesignated wells on a plate. A detection antibody (e.g. goat anti-humanIgG-HRP conjugate) is then added to each well followed by a chromagenicsubstrate (e.g. HRP). The plate is then read in ELISA plate reader at450 nM, and EC50 values are calculated. (It is understood, however, thatthe methods described here are generally applicable; they are notlimited to the production of antibodies that bind any particular antigenor class of antigens.)

Those of ordinary skill in the art will recognize that determiningaffinity is not always as simple as looking at a single figure. Sinceantibodies have two arms, their apparent affinity is usually much higherthan the intrinsic affinity between the variable region and the antigen(this is believed to be due to avidity). Intrinsic affinity can bemeasured using scFv or Fab fragments.

In another aspect, the present invention features bispecific moleculescomprising a humanized rabbit antibody, or a fragment thereof, of theinvention. An antibody of the invention, or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody of theinvention may be derivatized or linked to more than one other functionalmolecule to generate multispecific molecules that bind to more than twodifferent binding sites and/or target molecules; such multispecificmolecules are also intended to be encompassed by the term “bispecificmolecule” as used herein. To create a bispecific molecule of theinvention, an antibody of the invention can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other binding molecules, such as anotherantibody, antibody fragment, tumor specific or pathogen specificantigens, peptide or binding mimetic, such that a bispecific moleculeresults. Accordingly, the present invention includes bispecificmolecules comprising at least one first binding molecule havingspecificity for a first target and a second binding molecule havingspecificity for one or more additional target epitope.

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules of the invention are murine,chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities using methods known inthe art. For example, each binding specificity of the bispecificmolecule can be generated separately and then conjugated to one another.When the binding specificities are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (MA), flow cytometry based single cell sorting (e.g.FACS analysis), bioassay (e.g., growth inhibition), or Western Blotassay. Each of these assays generally detects the presence ofprotein-antibody complexes of particular interest by employing a labeledreagent (e.g., an antibody) specific for the complex of interest. Forexample, the antibody complexes can be detected using e.g., anenzyme-linked antibody or antibody fragment which recognizes andspecifically binds to the antibody-VEGF complexes. Alternatively, thecomplexes can be detected using any of a variety of other immunoassays.For example, the antibody can be radioactively labeled and used in aradioimmunoassay (RIA) (see, for example, Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986, which is incorporated byreference herein). The radioactive isotope can be detected by such meansas the use of a γ counter or a scintillation counter or byautoradiography.

In another aspect, the present invention features a humanized rabbitantibody, or a fragment thereof, conjugated to a therapeutic moiety,such as a cytotoxin, a drug (e.g., an immunosuppressant) or aradiotoxin. Such conjugates are referred to herein as“immunoconjugates”. Immunoconjugates that include one or more cytotoxinsare referred to as “immunotoxins.” A cytotoxin or cytotoxic agentincludes any agent that is detrimental to (e.g., kills) cells. Examplesinclude taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can beconjugated to an antibody of the invention include duocarmycins,calicheamicins, maytansines and auristatins, and derivatives thereof. Anexample of a calicheamicin antibody conjugate is commercially available(Mylotarg™; Wyeth-Ayerst).

Cytoxins can be conjugated to antibodies of the invention using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003)Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I.and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091;Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev.53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Method for preparing radioimmunconjugates areestablished in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (IDEC Pharmaceuticals) andBexxar™ (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the invention.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

In one aspect the invention provides pharmaceutical formulationscomprising humanized rabbit antibodies for the treatment disease. Theterm “pharmaceutical formulation” refers to preparations which are insuch form as to permit the biological activity of the antibody orantibody derivative to be unequivocally effective, and which contain noadditional components which are toxic to the subjects to which theformulation would be administered. “Pharmaceutically acceptable”excipients (vehicles, additives) are those which can reasonably beadministered to a subject mammal to provide an effective dose of theactive ingredient employed.

A “stable” formulation is one in which the antibody or antibodyderivative therein essentially retains its physical stability and/orchemical stability and/or biological activity upon storage. Variousanalytical techniques for measuring protein stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example.Stability can be measured at a selected temperature for a selected timeperiod. Preferably, the formulation is stable at room temperature (about30° C.) or at 40° C. for at least 1 month and/or stable at about 2-8° C.for at least 1 year for at least 2 years. Furthermore, the formulationis preferably stable following freezing (to, e.g., −70° C.) and thawingof the formulation.

An antibody or antibody derivative “retains its physical stability” in apharmaceutical formulation if it shows no signs of aggregation,precipitation and/or denaturation upon visual examination of colorand/or clarity, or as measured by UV light scattering or by sizeexclusion chromatography.

An antibody or antibody derivative “retains its chemical stability” in apharmaceutical formulation, if the chemical stability at a given time issuch that the protein is considered to still retain its biologicalactivity as defined below. Chemical stability can be assessed bydetecting and quantifying chemically altered forms of the protein.Chemical alteration may involve size modification (e.g. clipping) whichcan be evaluated using size exclusion chromatography, SDS-PAGE and/ormatrix-assisted laser desorption ionization/time-of-flight massspectrometry (MALDI/TOF MS), for example. Other types of chemicalalteration include charge alteration (e.g. occurring as a result ofdeamidation) which can be evaluated by ion-exchange chromatography, forexample.

An antibody or antibody derivative “retains its biological activity” ina pharmaceutical formulation, if the biological activity of the antibodyat a given time is within about 10% (within the errors of the assay) ofthe biological activity exhibited at the time the pharmaceuticalformulation was prepared as determined in an antigen binding assay, forexample. Other “biological activity” assays for antibodies areelaborated herein below.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example.

A “polyol” is a substance with multiple hydroxyl groups, and includessugars (reducing and non-reducing sugars), sugar alcohols and sugaracids. Preferred polyols herein have a molecular weight which is lessthan about 600 kD (e.g. in the range from about 120 to about 400 kD). A“reducing sugar” is one which contains a hemiacetal group that canreduce metal ions or react covalently with lysine and other amino groupsin proteins and a “non-reducing sugar” is one which does not have theseproperties of a reducing sugar. Examples of reducing sugars arefructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose and glucose. Non-reducing sugars include sucrose,trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol,erythritol, threitol, sorbitol and glycerol are examples of sugaralcohols. As to sugar acids, these include L-gluconate and metallicsalts thereof. Where it is desired that the formulation is freeze-thawstable, the polyol is preferably one which does not crystallize atfreezing temperatures (e.g. −20° C.) such that it destabilizes theantibody in the formulation. Non-reducing sugars such as sucrose andtrehalose are the preferred polyols herein, with trehalose beingpreferred over sucrose, because of the superior solution stability oftrehalose.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer of this invention has a pH in the range from about 4.5 to about6.0; preferably from about 4.8 to about 5.5; and most preferably has apH of about 5.0. Examples of buffers that will control the pH in thisrange include acetate (e.g. sodium acetate), succinate (such as sodiumsuccinate), gluconate, histidine, citrate and other organic acidbuffers. Where a freeze-thaw stable formulation is desired, the bufferis preferably not phosphate.

In a pharmacological sense, in the context of the present invention, a“therapeutically effective amount” of an antibody or antibody derivativerefers to an amount effective in the prevention or treatment of adisorder for the treatment of which the antibody or antibody derivativeis effective. A “disease/disorder” is any condition that would benefitfrom treatment with the antibody or antibody derivative. This includeschronic and acute disorders or diseases including those pathologicalconditions which predispose the mammal to the disorder in question.

A “preservative” is a compound which can be included in the formulationto essentially reduce bacterial action therein, thus facilitating theproduction of a multi-use formulation, for example. Examples ofpotential preservatives include octadecyldimethylbenzyl ammoniumchloride, hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofpreservatives include aromatic alcohols such as phenol, butyl and benzylalcohol, alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferredpreservative herein is benzyl alcohol.

The present invention also provides pharmaceutical compositionscomprising one or more antibodies or antibody derivative compounds,together with at least one physiologically acceptable carrier orexcipient. Pharmaceutical compositions may comprise, for example, one ormore of water, buffers (e.g., neutral buffered saline or phosphatebuffered saline), ethanol, mineral oil, vegetable oil,dimethylsulfoxide, carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, adjuvants, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione and/or preservatives. As noted above, other activeingredients may (but need not) be included in the pharmaceuticalcompositions provided herein.

A carrier is a substance that may be associated with an antibody orantibody derivative prior to administration to a patient, often for thepurpose of controlling stability or bioavailability of the compound.Carriers for use within such formulations are generally biocompatible,and may also be biodegradable. Carriers include, for example, monovalentor multivalent molecules such as serum albumin (e.g., human or bovine),egg albumin, peptides, polylysine and polysaccharides such asaminodextran and polyamidoamines. Carriers also include solid supportmaterials such as beads and microparticles comprising, for example,polylactate polyglycolate, poly(lactide-co-glycolide), polyacrylate,latex, starch, cellulose or dextran. A carrier may bear the compounds ina variety of ways, including covalent bonding (either directly or via alinker group), noncovalent interaction or admixture.

Pharmaceutical compositions may be formulated for any appropriate mannerof administration, including, for example, topical, oral, nasal, rectalor parenteral administration. In certain embodiments, compositions in aform suitable for oral use are preferred. Such forms include, forexample, pills, tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs. Within yet other embodiments, compositions providedherein may be formulated as a lyophilizate. The term parenteral as usedherein includes subcutaneous, intradermal, intravascular (e.g.,intravenous), intramuscular, spinal, intracranial, intrathecal andintraperitoneal injection, as well as any similar injection or infusiontechnique.

Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and may contain one or more agents, such as sweeteningagents, flavoring agents, coloring agent, and preserving agents in orderto provide appealing and palatable preparations. Tablets contain theactive ingredient in admixture with physiologically acceptableexcipients that are suitable for the manufacture of tablets. Suchexcipients include, for example, inert diluents (e.g., calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate), granulating and disintegrating agents (e.g., corn starch oralginic acid), binding agents (e.g., starch, gelatin or acacia) andlubricating agents (e.g., magnesium stearate, stearic acid or talc). Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monosterate or glyceryl distearatemay be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent(e.g., calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium (e.g., peanut oil, liquid paraffin or olive oil). Aqueoussuspensions contain the antibody or antibody derivative in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients include suspending agents (e.g., sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia);and dispersing or wetting agents (e.g., naturally-occurring phosphatidessuch as lecithin, condensation products of an alkylene oxide with fattyacids such as polyoxyethylene stearate, condensation products ofethylene oxide with long chain aliphatic alcohols such asheptadecaethyleneoxycetanol, condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides such as polyethylene sorbitan monooleate). Aqueoussuspensions may also comprise one or more preservatives, for exampleethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents, and one or more sweetening agents, such assucrose or saccharin. Syrups and elixirs may be formulated withsweetening agents, such as glycerol, propylene glycol, sorbitol, orsucrose. Such formulations may also comprise one or more demulcents,preservatives, flavoring agents, and/or coloring agents.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil (e.g., arachis oil, olive oil, sesame oil, or coconutoil) or in a mineral oil such as liquid paraffin. The oily suspensionsmay contain a thickening agent such as beeswax, hard paraffin, or cetylalcohol. Sweetening agents, such as those set forth above, and/orflavoring agents may be added to provide palatable oral preparations.Such suspensions may be preserved by the addition of an anti-oxidantsuch as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil (e.g., olive oil orarachis oil), a mineral oil (e.g., liquid paraffin), or a mixturethereof. Suitable emulsifying agents include naturally-occurring gums(e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides(e.g., soy bean, lecithin, and esters or partial esters derived fromfatty acids and hexitol), anhydrides (e.g., sorbitan monooleate), andcondensation products of partial esters derived from fatty acids andhexitol with ethylene oxide (e.g., polyoxyethylene sorbitan monoleate).An emulsion may also comprise one or more sweetening and/or flavoringagents.

The pharmaceutical composition may be prepared as a sterile injectibleaqueous or oleaginous suspension in which the modulator, depending onthe vehicle and concentration used, is either suspended or dissolved inthe vehicle. Such a composition may be formulated according to the knownart using suitable dispersing, wetting agents and/or suspending agentssuch as those mentioned above. Among the acceptable vehicles andsolvents that may be employed are water, 1,3-butanediol, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils may be employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectible compositions, and adjuvants such aslocal anesthetics, preservatives and/or buffering agents can bedissolved in the vehicle.

Pharmaceutical compositions may be formulated as sustained releaseformulations (i.e., a formulation such as a capsule that effects a slowrelease of modulator following administration). Such formulations maygenerally be prepared using well known technology and administered by,for example, oral, rectal, or subcutaneous implantation, or byimplantation at the desired target site. Carriers for use within suchformulations are biocompatible, and may also be biodegradable;preferably the formulation provides a relatively constant level ofmodulator release. The amount of an antibody or antibody derivativecontained within a sustained release formulation depends upon, forexample, the site of implantation, the rate and expected duration ofrelease and the nature of the disease/disorder to be treated orprevented.

Antibody or antibody derivatives provided herein are generallyadministered in an amount that achieves a concentration in a body fluid(e.g., blood, plasma, serum, CSF, synovial fluid, lymph, cellularinterstitial fluid, tears or urine) that is sufficient to detectablybind to a target such as e.g. VEGF and prevent or inhibit such targetmediated diseases/disorders, e.g. VEGF-mediated diseases/disorders. Adose is considered to be effective if it results in a discerniblepatient benefit as described herein. Preferred systemic doses range fromabout 0.1 mg to about 140 mg per kilogram of body weight per day (about0.5 mg to about 7 g per patient per day), with oral doses generallybeing about 5-20 fold higher than intravenous doses. The amount ofantibody or antibody derivative that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Dosage unitforms will generally contain between from about 1 mg to about 500 mg ofan active ingredient.

Pharmaceutical compositions may be packaged for treating conditionsresponsive to an antibody or antibody derivative directed e.g. to VEGF.Packaged pharmaceutical compositions may include a container holding aeffective amount of at least one antibody or antibody derivative asdescribed herein and instructions (e.g., labeling) indicating that thecontained composition is to be used for treating a disease/disorderresponsive to one antibody or antibody derivative followingadministration in the patient.

The antibodies or antibody derivatives of the present invention can alsobe chemically modified. Preferred modifying groups are polymers, forexample an optionally substituted straight or branched chain polyalkene,polyalkenylene, or polyoxyalkylene polymer or a branched or unbranchedpolysaccharide. Such effector group may increase the half-live of theantibody in vivo. Particular examples of synthetic polymers includeoptionally substituted straight or branched chain poly(ethyleneglycol)(PEG), poly(propyleneglycol), poly(vinylalcohol) or derivatives thereof.Particular naturally occurring polymers include lactose, amylose,dextran, glycogen or derivatives thereof. The size of the polymer may bevaried as desired, but will generally be in an average molecular weightrange from 500 Da to 50000 Da. For local application where the antibodyis designed to penetrate tissue, a preferred molecular weight of thepolymer is around 5000 Da. The polymer molecule can be attached to theantibody, in particular to the C-terminal end of the Fab fragment heavychain via a covalently linked hinge peptide as described in WO0194585.Regarding the attachment of PEG moieties, reference is made to“Poly(ethyleneglycol) Chemistry, Biotechnological and BiomedicalApplications”, 1992, J. Milton Harris (ed), Plenum Press, New York and“Bioconjugation Protein Coupling Techniques for the BiomedicalSciences”, 1998, M. Aslam and A. Dent, Grove Publishers, New York.

After preparation of the antibody or antibody derivative of interest asdescribed above, the pharmaceutical formulation comprising it isprepared. The antibody to be formulated has not been subjected to priorlyophilization and the formulation of interest herein is an aqueousformulation. Preferably the antibody or antibody derivative in theformulation is an antibody fragment, such as an scFv. Thetherapeutically effective amount of antibody present in the formulationis determined by taking into account the desired dose volumes andmode(s) of administration, for example. From about 0.1 mg/ml to about 50mg/ml, preferably from about 0.5 mg/ml to about 25 mg/ml and mostpreferably from about 2 mg/ml to about 10 mg/ml is an exemplary antibodyconcentration in the formulation.

An aqueous formulation is prepared comprising the antibody or antibodyderivative in a pH-buffered solution. The buffer of this invention has apH in the range from about 4.5 to about 6.0, preferably from about 4.8to about 5.5, and most preferably has a pH of about 5.0. Examples ofbuffers that will control the pH within this range include acetate (e.g.sodium acetate), succinate (such as sodium succinate), gluconate,histidine, citrate and other organic acid buffers. The bufferconcentration can be from about 1 mM to about 50 mM, preferably fromabout 5 mM to about 30 mM, depending, for example, on the buffer and thedesired isotonicity of the formulation. The preferred buffer is sodiumacetate (about 10 mM), pH 5.0.

A polyol, which acts as a tonicifier and may stabilize the antibody, isincluded in the formulation. In preferred embodiments, the formulationdoes not contain a tonicifying amount of a salt such as sodium chloride,as this may cause the antibody or antibody derivative to precipitateand/or may result in oxidation at low pH. In preferred embodiments, thepolyol is a non-reducing sugar, such as sucrose or trehalose. The polyolis added to the formulation in an amount which may vary with respect tothe desired isotonicity of the formulation. Preferably the aqueousformulation is isotonic, in which case suitable concentrations of thepolyol in the formulation are in the range from about 1% to about 15%w/v, preferably in the range from about 2% to about 10% whv, forexample. However, hypertonic or hypotonic formulations may also besuitable. The amount of polyol added may also alter with respect to themolecular weight of the polyol. For example, a lower amount of amonosaccharide (e.g. mannitol) may be added, compared to a disaccharide(such as trehalose).

A surfactant is also added to the antibody or antibody derivativeformulation. Exemplary surfactants include nonionic surfactants such aspolysorbates (e.g. polysorbates 20, 80 etc) or poloxamers (e.g.poloxamer 188). The amount of surfactant added is such that it reducesaggregation of the formulated antibody/antibody derivative and/orminimizes the formation of particulates in the formulation and/orreduces adsorption. For example, the surfactant may be present in theformulation in an amount from about 0.001% to about 0.5%, preferablyfrom about 0.005% to about 0.2% and most preferably from about 0.01% toabout 0.1%.

In one embodiment, the formulation contains the above-identified agents(i.e. antibody or antibody derivative, buffer, polyol and surfactant)and is essentially free of one or more preservatives, such as benzylalcohol, phenol, m-cresol, chlorobutanol and benzethonium Cl. In anotherembodiment, a preservative may be included in the formulation,particularly where the formulation is a multidose formulation. Theconcentration of preservative may be in the range from about 0.1% toabout 2%, most preferably from about 0.5% to about 1%. One or more otherpharmaceutically acceptable carriers, excipients or stabilizers such asthose described in Remington's Pharmaceutical Sciences 21st edition,Osol, A. Ed. (2006) may be included in the formulation provided thatthey do not adversely affect the desired characteristics of theformulation. Acceptable carriers, excipients or stabilizers arenon-toxic to recipients at the dosages and concentrations employed andinclude; additional buffering agents; co-solvents; antioxidantsincluding ascorbic acid and methionine; chelating agents such as EDTA;metal complexes (e.g. Zn-protein complexes); biodegradable polymers suchas polyesters; and/or salt-forming counterions such as sodium.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to, or following, preparation of the formulation.

The formulation is administered to a mammal in need of treatment withthe antibody, preferably a human, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. In preferred embodiments, the formulationis administered to the mammal by intravenous administration. For suchpurposes, the formulation may be injected using a syringe or via an IVline, for example.

The appropriate dosage (“therapeutically effective amount”) of theantibody will depend, for example, on the condition to be treated, theseverity and course of the condition, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, the type ofantibody used, and the discretion of the attending physician. Theantibody or antibody derivative is suitably administered to the patentat one time or over a series of treatments and may be administered tothe patent at any time from diagnosis onwards. The antibody or antibodyderivative may be administered as the sole treatment or in conjunctionwith other drugs or therapies useful in treating the condition inquestion.

As a general proposition, the therapeutically effective amount of theantibody or antibody derivative administered will be in the range ofabout 0.1 to about 50 mg/kg of patent body weight whether by one or moreadministrations, with the typical range of antibody used being about 0.3to about 20 mg/kg, more preferably about 0.3 to about 15 mg/kg,administered daily, for example. However, other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques.

In another embodiment of the invention, an article of manufacture isprovided comprising a container which holds the pharmaceuticalformulation of the present invention, preferably an aqueous formulation,and optionally provides instructions for its use. Suitable containersinclude, for example, bottles, vials and syringes. The container may beformed from a variety of materials such as glass or plastic. Anexemplary container is a 3-20 cc single use glass vial. Alternatively,for a multidose formulation, the container may be 3-100 cc glass vial.The container holds the formulation and the label on, or associatedwith, the container may indicate directions for use. The article ofmanufacture may further include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

In certain preferred embodiments, the article of manufacture comprises alyophilized immunobinder as described herein or generated by the methodsdescribed herein.

EXEMPLIFICATION

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference in their entireties.

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, immunology (especially, e.g.,antibody technology), and standard techniques of polypeptidepreparation. See, e.g., Sambrook, Fritsch and Maniatis, MolecularCloning: Cold Spring Harbor Laboratory Press (1989); AntibodyEngineering Protocols (Methods in Molecular Biology), 510, Paul, S.,Humana Pr (1996); Antibody Engineering: A Practical Approach (PracticalApproach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: ALaboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); andCurrent Protocols in Molecular Biology, eds. Ausubel et al., John Wiley& Sons (1992). Methods of grafting CDRs from rabbit and other non-humanmonoclonal antibodies onto selected human antibody frameworks isdescribed in detail above. Examples of such grafting experiments are setforth below.

For purposes of better understanding, grafts denominated “min” are thosewhere CDRs were grafted onto framework 1.4 or a variable domain thereof,whereas grafts named “max” are those where CDRs were grafted ontoframework 1.4 or a variable domain thereof and wherein the frameworkfurther comprises donor framework residues which interact with theantigen.

Example 1 Design of rFW1.4

1.1. Primary Sequence Analysis and Database Searching

1.1.1. Collection of Rabbit Immunoglobulin Sequences

Sequences of variable domains of rabbit mature antibodies and germlineswere collected from different open source databases (e.g. Kabat databaseand IMGT) and entered into a customized database as one letter codeamino acid sequences. For the entire analysis we used only the aminoacid portion corresponding to the V (variable) region. Sequences in theKDB database less than 70% complete or containing multiple undeterminedresidues in the framework regions were discarded. Sequences with morethan 95% identity to any other sequence within the database were alsoexcluded to avoid random noise in the analysis.

1.1.2. Alignments and Numbering of Rabbit Sequences

Rabbit antibody sequences were aligned using conventional sequencealignment tools based on the Needleman-Wunsch algorithm and Blossummatrices. The introduction of gaps and nomenclature of residue positionswere done following AHo's numbering system for immunoglobulin variabledomains (Honegger and Pluckthun, 2001). The Kabat numbering scheme wasalso applied in parallel since it is the most widely adopted standardfor numbering the residues in an antibody. Kabat numbering was assignedusing the SUBIM program. This program analyses variable regions of anantibody sequence and numbers the sequence according to the systemestablished by Kabat and co-workers (Deret et al 1995).

The definition of framework and CDR regions was done following the Kabatdefinition which is based on sequence variability and is the mostcommonly used. Nevertheless, CDR-H1 designation was a compromise betweendifferent definitions including, AbM's, kabat's, mean contact datagenerated by analysis of contacts between antibody and antigen of asubset of 3D complex structures (MacCallum et al., 1996) and Chotia'swhich is based on the location of the structural loop regions (describedabove and shown in FIG. 1).

1.1.3. Frequency and Conservation of Residue Positions

Amino acid sequence diversity was analyzed using a set of 423 rabbitsequences from the kabat database. The residue frequency, f(r), for eachposition, i, in the mature rabbit sequences was calculated by the numberof times that particular residue is observed within the data set dividedby the total number of sequences. The degree of conservation for eachposition, i, was calculated using the Simpson's index, which takes intoaccount the number of different amino acids present, as well as therelative abundance of each residue.

$D = \frac{\sum_{i = 1}^{r}{n\left( {n - 1} \right)}}{N\left( {N - 1} \right)}$

where: N Total number of amino acids, r is the number of different aminoacids present at each position and n is the number of residues of aparticular amino acid type.

1.1.4. Lineage Analysis of the Rabbit V Region

Phylogeny analysis tools were used to study the rabbit repertoire. Aminoacids sequences of the V region were clustered using both cluster andtopological algorithms. The distance matrix was calculated for the wholearray and used as indication of the germline usage. Consensus sequenceof each cluster was calculated and the nearest rabbit germline sequencecounterpart identified. Also the overall consensus sequence was derivedfor the whole set of sequences.

1.1.5. Assignment of Human Subgroup.

For each rabbit representative sequence of the different clusters, themost homologous human sub-group was identified using an EXCELimplementation of sequence analysis algorithms and classificationmethods based on analysis of the human antibody repertoire (Knappik etal., 2000).

1.2. Design of the Human Acceptor Framework

With the blueprint of the rabbit repertoire from the sequence analysisdescribed above, residues in the framework generally involved in thepositioning of rabbit CDRs were identified. Among the frameworks havinghigh homology relative to the rabbit repertoire and the respectiveclusters, one having good biophysical properties was selected from apool of fully human sequences. The selected framework to serve asacceptor framework belongs to variable light chain subgroup kappa 1 andheavy variable chain subgroup III with the ESBATech's ID KI 27, a43correspondingly. This stable and soluble antibody framework has beenidentified by screening of a human spleen scFv library using ayeast-based screening method named “Quality Control” system (Auf derMaur et al., 2004) and was designated “FW1.4”. Although the stable andsoluble framework sequence FW1.4 exhibits high homology, it was not themost homologous sequence available. The identified residues wereincorporated in said acceptor framework to generate rFW1.4.

With the information for amino acid sequence diversity, germline usageand the structural features of the rabbit antibodies, we analyzed theFW1.4 for compatibility of residue positions required to preserve theCDR conformation in the new human framework. We examined the variableregions of FW 1.4 for compatibility of the following characteristics:

i. Residues that are part of the canonical sequences for loopstructures.

ii. Framework residues located at the VL/VH interface.

iii. The platform of residues directly underneath the CDRs

iv. Upper and lower core residues

v. Framework residues defining the subtype

1.3. Grafting of Rabbit CDRs

Grafts were generated by simply combining the CDR sequences (accordingabove definition) from one antibody with the framework sequence of FW1.4 or the rFW1.4. Residues potentially involved in binding wereidentified.

For each rabbit variable domain sequence, the nearest rabbit germlinecounterpart was identified. If the closest germline could not beestablished, the sequence was compared against the subgroup consensus orthe consensus of rabbit sequences with a high percentage of similarity.Rare framework residues were considered as possible result of somatichypermutation and therefore playing a role in binding. Consequently,such residues were grafted onto the acceptor framework.

1.4 Results

By analyzing the rabbit antibody repertoire in terms of structure, aminoacid sequence diversity and germline usage, 5 residue positions in thelight chain of FW1.4 were found which were modified to maintain loopconformation of rabbit CDRs. These positions are highly conserved inrabbit antibodies. The consensus residue for these 5 positions wasdeduced from the rabbit repertoire and introduced into the humanacceptor framework 1.4. With the modification of these conservedpositions, said framework became virtually compatible with all sixcomplementarity determining regions (CDRs) of any rabbit CDRs. Themaster rFW1.4 containing different rabbit CDRs is well expressed andgood produced contrary to the wild type single chains. 16 membersderived from the combination of this framework and rabbit CDRs werecreated detailed characterization showed functionality.

Example 2 B Cell Screening System

A FACS (flow cytometry based single cell sorting)-based screening systemhas been established at ESBATech in order to select B cells that bind toa target of interest via their B cell receptors (BCR). One target wasfor example a soluble protein, namely a single-chain antibody (ESBA903)labeled with a fluorescent dye (PE and PerCP). Lymphocyte suspension wasprepared from the spleen of rabbits immunized with the recombinanttarget. Cells were then incubated with PE and PerCP labeled ESBA903 aswell as with antibodies specific for IgG (APC-labeled) or IgM (FITClabeled). ESBA903 positive B-cells that express IgG but not IgM on theirsurface were sorted in 96-well plates (FIG. 3; table 2). By means of athymoma helper cell line (EL4-B5: see Zubler et al, 1985, J. Immunol,134(6): 3662-3668), selected B cells proliferated, differentiated intoplasma cells and secreted antibodies. The affinity of these IgGs for thetarget was verified by ELISA and Biacore measurements. Kineticparameters are depicted in table 1 for seven selected clones. Theseclones, from a pool of ˜200 sorted cells, show binding affinities in thelow nanomolar to picomolar range. Finally, mRNA was isolated from 6clones of interest and CDRs were grafted on the single-chain frameworkFW1.4.

TABLE 1 Kinetic values for 7 B cell culture supernatants. B-cell cloneka [Ms⁻¹] kd [s⁻¹] K_(D) [M] SG2 2.91E+06 2.95E−04 1.01E−10 SE113.63E+05 3.81E−04 1.05E−09 2E-03 8.34E+05 3.53E−04 4.23E−10 9E-038.66E+05 6.47E−04 7.47E−10 7D-03 3.97E+05 3.04E−04 7.65E−10 12B-021.08E+06 1.10E−04 1.01E−10

TABLE 2 Sorting statistics. Population #events % parent % total Allevents 100.000 #### 100.0 Lymphocytes 86.585 86.6 86.6 SingleLymphocytes 1 86.013 99.3 86.0 Single Lymphocytes 2 85.523 99.4 85.5Memory B cells? 5.450 6.4 5.4 Sorted cells 16 0.3 0.0 903-binding cells160 2.9 0.2

Example 3 Detection of the Interaction Between Beads Coated withAnti-TNFalpha Antibody and CHO Cells Expressing Membrane-Bound TNFalpha

In order to evaluate whether or not the high pressure in flow-cytometrystream breaks non covalent binding between two cells, the followingexperiment was performed.

CHO cells stably transfected with membrane-bound TNFalpha (B-220 cells)were incubated with beads coated with a PE-labeled anti-TNFalphaantibody. In this set-up the beads mimic memory B cells (they have moreor less the same size). As negative controls, non-transfected CHO cellswere used, as well as beads coated with an APC labeled unrelatedantibody (anti-CD19). After 2 hours incubation at 4° C. with agitation,the cell-bead suspension was analyzed by FACS (using a 130 um nozzle).FIG. 4 shows that a specific binding between anti-TNFalpha □ beads andTNFalpha-transfected CHO cells is clearly detectable with FACS. Indeed,in this sample (upper panel) about two thirds of the beads are bound tocells (585 bound against 267 unbound). In contrast, in the controlsamples (middle and lower panels), almost no bead binds to CHO cells.Further, both bead populations (anti-TNFalpha-PE and anti-CD19-APC) weremixed together with TNFalpha-transfected CHO cells. FIG. 5 and table 4shows that about half of the anti-TNFalpha beads bind to CHO cells,whereas the vast majority of the anti-CD19 beads stay unbound. Thepercentage of beads binding to the cell in each sample is detailed intable 5. Thus, the demonstration is made that the specific selection ofsingle B cells that bind to an integral membrane target protein throughtheir B cell receptor is possible using flow-cytometry.

TABLE 3a Sorting statistics (see also FIG. 4a) Population # Events %Parent % Total All events 10.000 ### 100.0 P1 9.692 96.9 96.9 P3 585 6.05.9 P4 1 0.0 0.0 P2 267 2.7 2.7

TABLE 3b Sorting statistics (see also FIG. 4b) Population # Events %Parent % Total All events 10.000 ### 100.0 P1 9.399 94.0 94.0 P3 3 0.00.0 P4 6 0.1 0.1 P2 550 5.6 5.6

TABLE 3c Sorting statistics (see also FIG. 4c) Population # Events %Parent % Total All events 10.000 ### 100.0 P1 9.001 90.0 90.0 P3 13 0.10.1 P4 7 0.1 0.1 P2 811 8.1 8.1

TABLE 4 Sorting statistics (see also FIG. 5) Population # Events %Parent % Total All events 10.000 ### 100.0 P1 9.096 91.0 91.0 P3 401 4.44.0 P4 2 0.0 0.0 P2 856 8.6 8.6

TABLE 5 Percentage of beads bound to CHO cells in each sample Cells mAbon beads % bound beads Sample 1 CHO-TNFα (B220) anti-TNFα 68.0 Sample 2CHO-TNFα (B220) anti-CD19 0.9 Sample 3 CHO wt anti-TNFα 1.5 Sample 4CHO-TNFα (B220) anti-TNFα 47.0 anti-CD19 0.4

Example 4 CDR Grafting and Functional Humanization of Anti-TNFα RabbitDonor Antibodies

Four anti-TNFα rabbit antibodies “Rabmabs” (EPI-1, EPI-15, EP-34, EP-35and EP-42) were selected for CDR grafting. The general experimentalscheme for the CDR grafting, humanization, and preliminarycharacterization of humanized rabbit donor antibodies was done asoutlined in the description. Unlike traditional humanization methodswhich employ the human antibody acceptor framework that shares thegreatest sequence homology with the non-human donor antibody, the rabbitCDRs were grafted into a human framework (FW 1.4) that was preselectedfor desirable functional properties (solubility and stability) using aQuality Control assay (WO0148017). These stable and soluble frameworksequence exhibited high homology with the RabMabs.

CDR grafts were generated for each of the RabMabs using the methodologydescribed herein. In “Min” grafts, only the rabbit CDRs weretransplanted from the VL and VH domains of the rabbit donor antibody tothe human acceptor framework FW1.4. “Max” grafts refer to the graftingof the rabbit CDRs to rFW1.4.

The scFvs described and characterized herein were produced as follows.The humanized VL sequences were connected to humanized VH sequences viathe linker of SEQ ID NO:8 to yield an scFv of the following orientation:NH₂-VL-linker-VH-COOH. In many cases DNA sequences encoding for thevarious scFvs were de novo synthesized at the service providerEntelechon GmbH. The resulting DNA inserts were cloned into thebacterial expression vector pGMP002 via NcoI and HindIII restrictionsites introduced at the 5′ and 3′ end of the scFv DNA sequence,respectively. Between the DNA sequence of the VL domain and the VHdomain, a BamHI restriction site is located. In some cases the scFvencoding DNA was not de novo synthesized, but the scFv expressingconstructs were cloned by domain shuffling. Accordingly, the VL domainswere excised and introduced into the new constructs via NcoI and BamHIrestriction sites, the VH domains via BamHI and HindIII restrictionsites. In other cases, point mutations were introduced into the VHand/or VL domain using state of the art assembling PCR methods. Thecloning of GMP002 is described in Example 1 of WO2008006235. Theproduction of the scFvs was done analogue as for ESBA105 as described inExample 1 of WO2008006235.

Table 3 depicts a summary of the detailed characterization data for thefour rabbit monoclonals (EP6, EP19, EP34, EP35 and EP43) and their CDRgrafted variants. Although the CDR grafts exhibited a broad range ofactivities in BIACore binding assays and L929, TNFα-mediatedcytotoxocity assays, 3 of the 4 maximal (“max”) grafts exhibitedtherapeutically relevant activities. EP43max exhibited the mostfavorable binding affinity (Kd of 0.25 nM) and an excellent EC50 in thecytotoxicity assay. This data show that FW1.4 (SEQ ID No: 1 and 2) is anexemplary soluble and stable human acceptor framework region forgrafting rabbit CDRs.

Potency Assay

The neutralizing activity of anti-TNFα binders was assessed in a L929TNFα-mediated cytotoxicity assay. Toxicity of Mouse L929 fibroblastcells treated with Actinomycin was induced with recombinant human TNF(hTNF). 90% of maximal hTNF-induced cytoxicity was determined to be at aTNF concentration of 1000 pg/ml. All L929 cells were cultured in RPMI1640 with phenolred, with L-Glutamine medium supplemented with fetalcalf serum (10% v/v). The neutralizing activity of anti-TNFα binders wasassessed in RPMI 1640 without phenolred and 5% fetal calf serum.Different concentrations (0-374 ng/mL) of anti-TNF binders are added toL929 cells in presence of 1000 pg/ml hTNF in order to determine theconcentration at which the antagonistic effect reaches half-maximalinhibition (EC50%) The dose response curve was fitted with nonlinearsigmoidal regression with variable slope and the EC50 was calculated.

Biacore Binding Analysis of Anti-TNF scFvs

For binding affinity measurements, surface Plasmon resonancemeasurements with BIAcore™-T100 were employed using a NTA sensor chipand His-tagged TNF (produced at ESBATech). The surface of the NTA sensorchip consists of a carboxymethylated dextran matrix pre-immobilized withnitrilotriacetic acid (NTA) for capture of histidine tagged moleculesvia Ni2+NTA chelation. Human TNFα N-his trimers (5 nM) are captured bythe nickel via their N-terminal his-tags and ESBA105 (analyte) isinjected at several concentrations ranging from 30 nM to 0.014 nM in 3fold serial dilution steps. At the regeneration step, the complex formedby nickel, ligand and analyte is washed away. This allows the use of thesame regeneration conditions for different samples. The response signalis generated by surface Plasmon resonance (SPR) technology and measuredin resonance units (RU). All the measurements are performed at 25° C.Sensorgrams were generated for each anti-TNF scFv sample after in-linereference cell correction followed by buffer sample subtraction. Theapparent dissociation rate constant (k_(d)), the apparent associationrate constant (k_(a)) and the apparent dissociation equilibrium constant(K_(D)) were calculated using one-to-one Langmuir binding model withBIAcore T100 evaluation Software version 1.1.

TABLE 3 TNFalpha second generation of binders Description ID L929* konkoff K_(D) FT-IR TM ° C. RF yield** EP1_min 1071 ND*** — — — — 2 EP6_min673 ND*** 4.67E+04 4.94E−03 1.06E−07 50.2 35 EP15_min 1073 ND***1.57E+05 4.10E−02 2.62E−07 — 41.5 EP19_min 616 ND*** — — — — — EP34_min643 ND*** — — — — — EP35_min 1075 ND*** — — — — 1 EP42_min 1076 ND***1.42E+05 8.35E−03 5.87E−08 — 3 EP43_min 705 ND*** 5.38E+03 2.98E−025.54E−06 70.2 30.0 EP1_max 1072 ND*** 1.11E+04 6.30E−04 5.69E−08 — 44EP6_max 674 1.1 2.84E+05 1.45E−04 5.12E−10 48.1 12 EP15_max 1074 0.391.53E+06 2.26E−03 1.48E−09 68.6 57.8 EP19_max 1007 0.6 2.25E+04 6.54E−052.91E−09 53.5 52 EP34_max 791 10.5 5.86E+05 1.68E−05 2.86E−11 72.4 4.05EP35_max 1089 5.20 7.72E+05 1.50E−04 1.94E−10 — 0.66 EP42_max 1077 ND***1.21E+05 4.19E−04 3.46E−09 — 47.6 EP43_max 676 6.4 1.78E+05 4.48E−052.51E−10 74.3 21.73 EP34min_C-His 790 0.2 EP19max_C-His 789 1.9 *L929[EC50-E105/EC50-X], compared in mass units [ng/ml] relative to theperformance of ESBA105(WO06/131013) **(mg/L refolding solution); ***NotDetermined

Example 5 CDR Grafting and Functional Humanization of Anti-VEGF RabbitDonor Antibodies

Eight anti-VEGF Rabmabs (375, 435, 509, 511, 534, 567, 578 and 610) wereselected for CDR grafting. Unlike traditional humanization methods whichemploy the human antibody acceptor framework that shares the greatestsequence homology with the non-human donor antibody, the rabbit CDRswere grafted into a human acceptor framework FW1.4 (SEQ ID No: 1 and 2)that was preselected for desirable functional properties (solubility andstability) using a Quality Control assay (WO0148017).

A number of CDR grafts were generated for each of the RabMabs (rabbitantibodies) using the methodology described herein (see Example 4).“Min” grafts comprised a minimal graft wherein only the rabbit CDRs weretransplanted from the VL and VH domains of the rabbit donor antibody tothe human acceptor framework FW1.4 (SEQ ID No: 1). “Max” graftscomprised not only the rabbit CDRs for the VL and VH, but also someadditional framework residues from the rabbit donor that were predictedto be important for antigen binding. In the case of 578max, the heavychain variable domain framework region of FW1.4 has additional aminoacid alterations at Kabat positions 23H, 49H, 73H, 78H, and 94H.

Table 4 shows a summary of the detailed characterization data for the“Min” and “Max” CDR grafted variants. Their potency as VEGF inhibitors,which is measured using VEGFR competition ELISA and/or HUVEC assay aredescribed. This data shows that FW1.4 (SEQ ID No: 1 and 2) is anexemplary soluble and stable human acceptor framework region forgrafting rabbit CDRs.

Biacore Binding Analysis of Anti-VEGF scFvs

The Biacore-binding ability of scFvs was tested and the binding affinitywas measured using the exemplary surface plasmon resonance method withBIAcore™-T100. The VEGF proteins, tested for binding by these scFvcandidates, in this example and later examples include purifiedEscherichia coli-expressed recombinant human VEGF₁₆₅ (PeproTech ECLtd.), recombinant human VEGF₁₂₁ (PeproTech EC Ltd.), recombinant humanVEGF₁₁₀ (ESBATech AG), recombinant murine VEGF₁₆₄ (PeproTech EC Ltd.),recombinant rat VEGF₁₆₄ (Biovision), recombinant rabbit VEGF₁₁₀(ESBATech AG), and recombinant human PLGF (PeproTech EC Ltd.). For thesurface plasmon resonance experiment, carboxymethylated dextranbiosensor chips (CM4, GE Healthcare) were activated withN-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride andN-hydroxysuccinimide according to the supplier's instructions. Each ofthe 6 different VEGF forms, as exemplified above, was coupled to 1 ofthe 4 different flow cells on a CM4 sensor chip using a standardamine-coupling procedure. The range of responses obtained with theseimmobilized VEGF molecules after coupling and blocking were ˜250-500response units (RU) for hVEGF₁₆₅, ˜200 RU for hVEGF₁₁₀, hVEGF₁₂₁, murineVEGF₁₆₄, rat VEGF₁₆₄ and rabbit VEGF₁₁₀ and ˜400 RU for PLGF. The 4thflow cell of each chip was treated similarly except no proteins wereimmobilized prior to blocking, and the flow cell was used as in-linereference. Various concentrations of anti-VEGF scFvs (e.g., 90 nM, 30nM, 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM and 0.04 nM) in HBS-EPbuffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactantP20) were injected into the flow cells at a flow rate of 30 μl/min for 5min. Dissociation of the anti-VEGF scFv from the VEGF on the CM4 chipwas allowed to proceed for 10 min at 25° C. Sensorgrams were generatedfor each anti-VEGF scFv sample after in-line reference cell correctionfollowed by buffer sample subtraction. The apparent dissociation rateconstant (k_(d)), the apparent association rate constant (k_(a)) and theapparent dissociation equilibrium constant (K_(D)) were calculated usingone-to-one Langmuir binding model with BIAcore T100 evaluation Softwareversion 1.1.

HUVEC Assay of VEGF Inhibition

The HUVEC assay is a method to measure the potency of the disclosedanti-VEGF scFv candidates as VEGF inhibitors.

Human umbilical vein endothelial cells (HUVECs) (Promocell), pooled fromseveral donors, were used at passage 2 to passage 14. Cells were seededat 1000 cells/well in 50 μl complete endothelial cell growth medium(ECGM) (Promocell), that contained 0.4% ECGS/H, 2% Fetal Calf Serum, 0.1ng/ml Epidermal Growth Factor, 1 μg/ml Hydrocortison, 1 ng/ml basicFibroblast Factor and 1% penicillin/streptomycin (Gibco). 7 to 8 hlater, 50 μl starving medium (ECGM without supplements containing 0.5%heat inactivated FCS and 1% penicillin/streptomycin) was added to thecells and the cells were starved for 15 to 16 hours. 3 fold Serialdilutions of anti-VEGF scFvs (0.023-150 nM) and one of thefollowing—recombinant human VEGF₁₆₅ (0.08 nM), recombinant mouse VEGF₁₆₄(0.08 nM), or recombinant rat VEGF₁₆₄ (0.3 nM)-were prepared in starvingmedium and preincubated for 30-60 min at room temperature. The differentconcentrations of VEGFs were used to compensate for their differentrelative biological activities. Concentrations that stimulate submaximalVEGF induced proliferation (EC₉₀) were used. 100 μl of the mixtures wereadded to the 96-well tissue-culture plates containing the HUVECsuspension and incubated for 4 days in a 37° C./5% CO₂ humifiedincubator. Proliferation of HUVECs was assessed by measuring absorbanceat 450 nm (620 nm used as reference wavelength) after addition of 20μl/well WST-1 cell proliferation reagent (Roche) using a Sunrisemicroplate reader (Tecan). Data were analyzed using a 4-parameterlogistic curve-fit, and the concentration of anti-VEGF scFvs required toinhibit HUVEC proliferation by 50% (EC₅₀) was derived from inhibitioncurves.

TABLE 4 Rel. activity hVEGR2 comp. Rel. activity hVEGR1 comp. BiacoreMeasurements ELISA ELISA hVEGF₁₆₅ ID Protein Nr. (EC50_(Luc)[nM]/EC50_(test) [nM]) (EC50_(Luc) [nM]/EC50_(test) [nM]) ka (1/Ms) kd(1/s) KD (M) 375-min 857 0.3 ND 9.27E+05 5.01E−03 5.41E−09 375-max 8730.6 ND 2.44E+06 6.55E−03 2.68E−09 375-max C-His 877 0.4 ND 2.93E+058.75E−04 2.98E−09 509-min 854 1.0 2.9 6.23E+05 1.14E−03 1.82E−09 509-max855 4.1 13 2.26E+06 2.72E−03 1.21E−09 509-maxII 856 0.6 0.09 8.38E+052.82E−03 3.37E−09 511-min 801 4.9 0.7 5.05E+05 1.28E−03 2.53E−09 511-max802 8.7 8 6.59E+05 4.40E−05 6.67E−11 534-min C-His 807 0.1 ND 2.71E+059.21E−03 3.41E−08 534-max 793 1.1 ND 1.88E+06 1.73E−02 9.21E−09 567-min884 9.7 57 2.01E+06 4.61E−04 2.30E−10 567-max 874 4.1 15.7/54.5 1.20E+062.26E−04 1.88E−10 578-min 820 4.1 4.8 1.14E+06 1.03E−02 9.01E−09 578-max821 9.6 35.5/51.6 7.00E+05 3.07E−04 4.39E−10 610-min 882 0.1 ND 2.51E+052.65E−03 1.06E−08 610-max 883 0.4 ND 5.09E+05 6.01E−04 1.18E−09 435-min944 ND ND ND ND ND 435-max 945 7.6 ND 1.67E+05 7.55E−04 4.53E−09

EQUIVALENTS

Numerous modifications and alternative embodiments of the presentinvention will be apparent to those skilled in the art in view of theforegoing description. Accordingly, this description is to be construedas illustrative only and is for the purpose of teaching those skilled inthe art the best mode for carrying out the present invention. Details ofthe structure may vary substantially without departing from the spiritof the invention, and exclusive use of all modifications that comewithin the scope of the appended claims is reserved. It is intended thatthe present invention be limited only to the extent required by theappended claims and the applicable rules of law.

All literature and similar material cited in this application,including, patents, patent applications, articles, books, treatises,dissertations, web pages, figures and/or appendices, regardless of theformat of such literature and similar materials, are expresslyincorporated by reference in their entirety. In the event that one ormore of the incorporated literature and similar materials differs fromor contradicts this application, including defined terms, term usage,described techniques, or the like, this application controls.

1. An immunobinder comprising: (i) light chain CDR1, CDR2 and CDR3 froma donor lagomorph immunobinder and heavy chain CDR1, CDR2 and CDR3 froma donor lagomorph immunobinder; (ii) a variable human light chainframework; and (iii) a variable human heavy chain framework that is atleast 85% identical to the sequence of SEQ ID NO: 4 and comprises athreonine (T) at position 24, alanine (A) or glycine (G) at position 56,threonine (T) or asparagine (N) at position 84, and leucine (L) orvaline (V) at position 89 (AHo numbering).
 2. The immunobinder of claim1, wherein the amino acid sequence of the variable heavy chain frameworkcomprises threonine (T) at position 24, glycine (G) at position 56,threonine (T) at position 84, valine (V) at position 89, and arginine(R) at position 108 (AHo numbering).
 3. The immunobinder of claim 1,wherein said variable heavy chain framework comprises glycine (G) atposition 56 (AHo numbering).
 4. The immunobinder of claim 1, whereinsaid variable heavy chain framework comprises threonine (T) at position84 (AHo numbering).
 5. The immunobinder of claim 1, wherein saidvariable heavy chain framework comprises valine (V) at position 89 (AHonumbering).
 6. The immunobinder of claim 1, wherein said variable heavychain framework comprises arginine (R) at position 108 (AHo numbering).7. The immunobinder of claim 1, wherein said variable heavy chainframework further comprises at least one of the following amino acids:Serine (S) at position 12; Serine (S) or Threonine (T) at position 103;and Serine (S) or Threonine (T) at position 144 (AHo numbering).
 8. Theimmunobinder of claim 1, wherein said variable heavy chain frameworkfurther comprises Glycine (G) at position 141 (AHo numbering).
 9. Theimmunobinder of claim 1, wherein said variable light chain frameworkcomprises an amino acid sequence that is at least 85% identical to thesequence of SEQ ID NO.
 2. 10. The immunobinder of claim 9, wherein saidvariable light chain framework comprises threonine (T) at position 87(AHo numbering).
 11. The immunobinder of claim 9, wherein said variableheavy chain framework and said variable light chain framework are linkedvia a linker sequence that comprises SEQ ID NO.
 8. 12. The immunobinderof claim 9 comprising an amino acid sequence that is at least 85%identical to the sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.13. The immunobinder of claim 9, wherein said variable light chainframework further comprises at least one of the following amino acids:glutamic acid (E) at position 1, valine (V) at position 3, leucine (L)at position 4, Serine (S) at position 10, Arginine (R) at position 47,Serine (S) at position 57, phenylalanine (F) at position 91, and Valine(V) at position 103 (AHo numbering).
 14. The immunobinder of claim 1,wherein said variable heavy chain framework further comprises a valine(V) at position 25 and/or a lysine (K) at position 82 (AHo numbering).15. The immunobinder of claim 1, wherein said immunobinder is an scFv.16. A pharmaceutical composition comprising the immunobinder of claim 1and a pharmaceutically acceptable excipient.
 17. A bispecific moleculecomprising the immunobinder of claim
 1. 18. The bispecific molecule ofclaim 17, which binds to at least two different binding sites or targetmolecules.
 19. The bispecific molecule of claim 17, wherein theimmunobinder is linked to an antibody, antibody fragment (including aFab, Fab′, F(ab′)₂, Fv or single chain Fv), tumor specific or pathogenspecific antigens, peptide or a binding mimetic.
 20. A method ofhumanizing a rabbit immunobinder, the rabbit immunobinder comprisingheavy chain CDR H1, CDR H2 and CDR H3 sequences and light chain CDR L1,CDR L2 and CDR L3 sequences, the method comprising: (i) grafting therabbit CDR H1, CDR H2 and CDR H3 sequences into a human heavy chainacceptor framework that is at least 85% identical to the sequence of SEQID NO: 4 and that comprises a threonine (T) at position 24 (AHonumbering), alanine (A) or glycine (G) at position 56, threonine (T) orasparagine (N) at position 84, and leucine (L) or valine (V) at position89 (AHo numbering), and (ii) grafting the rabbit CDR L1, CDR L2 and CDRL3 sequences into a human light chain acceptor framework, the humanlight chain framework having at least 85% identity to SEQ ID NO:2. 21.The method of claim 20, wherein the amino acid sequence of the variableheavy chain framework further comprises at least three of the followingamino acids: alanine (A) or glycine (G) at position 56, threonine (T) atposition 84, valine (V) at position 89, and arginine (R) at position 108(AHo numbering).